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      Silencing Mutant Ataxin-3 Rescues Motor Deficits and Neuropathology in Machado-Joseph Disease Transgenic Mice

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          Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.

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          Most cited references 35

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          Reversal of neuropathology and motor dysfunction in a conditional model of Huntington's disease.

          Neurodegenerative disorders like Huntington's disease (HD) are characterized by progressive and putative irreversible clinical and neuropathological symptoms, including neuronal protein aggregates. Conditional transgenic models of neurodegenerative diseases therefore could be a powerful means to explore the relationship between mutant protein expression and progression of the disease. We have created a conditional model of HD by using the tet-regulatable system. Mice expressing a mutated huntingtin fragment demonstrate neuronal inclusions, characteristic neuropathology, and progressive motor dysfunction. Blockade of expression in symptomatic mice leads to a disappearance of inclusions and an amelioration of the behavioral phenotype. We thus demonstrate that a continuous influx of the mutant protein is required to maintain inclusions and symptoms, raising the possibility that HD may be reversible.
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            SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein.

            Genetic etiologies of at least 20% of autosomal dominant cerebellar ataxias (ADCAs) have yet to be clarified. We identified a novel spinocerebellar ataxia (SCA) form in four Japanese pedigrees which is caused by an abnormal CAG expansion in the TATA-binding protein (TBP) gene, a general transcription initiation factor. Consequently, it has been added to the group of polyglutamine diseases. This abnormal expansion of glutamine tracts in TBP bears 47--55 repeats, whereas the normal repeat number ranges from 29 to 42. Immunocytochemical examination of a postmortem brain which carried 48 CAG repeats detected neuronal intranuclear inclusion bodies that stained with anti-ubiquitin antibody, anti-TBP antibody and with the 1C2 antibody that recognizes specifically expanded pathological polyglutamine tracts. We therefore propose that this new disease be called SCA17 (TBP disease).
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              Allele-specific silencing of dominant disease genes.

              Small interfering RNA (siRNA) holds therapeutic promise for silencing dominantly acting disease genes, particularly if mutant alleles can be targeted selectively. In mammalian cell models we demonstrate that allele-specific silencing of disease genes with siRNA can be achieved by targeting either a linked single-nucleotide polymorphism (SNP) or the disease mutation directly. For a polyglutamine neurodegenerative disorder in which we first determined that selective targeting of the disease-causing CAG repeat is not possible, we took advantage of an associated SNP to generate siRNA that exclusively silenced the mutant Machado-Joseph disease/spinocerebellar ataxia type 3 allele while sparing expression of the WT allele. Allele-specific suppression was accomplished with all three approaches currently used to deliver siRNA: in vitro-synthesized duplexes as well as plasmid and viral expression of short hairpin RNA. We further optimized siRNA to specifically target a missense Tau mutation, V337M, that causes frontotemporal dementia. These studies establish that siRNA can be engineered to silence disease genes differing by a single nucleotide and highlight a key role for SNPs in extending the utility of siRNA in dominantly inherited disorders.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                22 January 2013
                : 8
                : 1
                [1 ]CNC - Center for Neurosciences & Cell Biology, University of Coimbra, Coimbra, Portugal
                [2 ]Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
                [3 ]Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal
                [4 ]Department of Neurophysiology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan
                [5 ]Lausanne University Hospital, Department of Clinical Neurosciences, Laboratory of Cellular and Molecular Neurotherapies, Lausanne, Switzerland
                Dulbecco Telethon Institute and Mario Negri Institute for Pharmacological Research, Italy
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Financial support and administrative support: LPdA.. Conceived and designed the experiments: CN INF LPdA. Performed the experiments: CN INF IO DA. Analyzed the data: CN INF HH ND LPdA. Contributed reagents/materials/analysis tools: HH ND LPdA. Wrote the paper: CN LPdA.


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 11
                This work was supported by the Portuguese Foundation for Science and Technology (grants PTDC/SAU-NEU/099307/2008 and PTDC/SAU-FAR/116535/2010 and fellowships to CN, INF, IO and DA), the Center for Science and Technology of Madeira (CN), the National Ataxia Foundation and the Richard Chin and Lily Lock Machado-Joseph disease Research Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Genetic Engineering
                Human Genetics
                Autosomal Dominant
                Gene Therapy
                Vector Biology
                Viral Vectors
                Molecular Cell Biology
                Gene Expression
                RNA interference
                Nucleic Acids
                RNA interference
                Molecular Neuroscience
                Neurobiology of Disease and Regeneration
                Cerebellar Disorders
                Movement Disorders
                Neurodegenerative Diseases



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