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      RNA Interference Mitigates Motor and Neuropathological Deficits in a Cerebellar Mouse Model of Machado-Joseph Disease

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          Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.

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          Most cited references 34

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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.
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            Fourteen and counting: unraveling trinucleotide repeat diseases.

            The pathological expansion of unstable trinucleotide repeats currently is known to cause 14 neurological diseases. Over the past several years, researchers have concentrated on the challenging task of identifying the mechanism by which the expanded trinucleotide repeat leads to abnormal cellular function. As a consequence, the trinucleotide repeat field has grown dramatically since the initial discovery of dynamic mutations less than a decade ago. Trinucleotide repeat expansions may prove to cause pathology through a variety of mechanisms including interference with DNA structure, transcription, RNA-protein interaction and altered protein conformations/interactions. The goal of this review is to provide a brief description of the genes harboring expanded repeats, coupled with new insights into the molecular pathways most likely to be disrupted by these expansions. Data from studies of patient material, cell culture and animal models demonstrate the complexity of the pathogenic mechanisms in each of the diseases.
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              Overexpression of the autophagic beclin-1 protein clears mutant ataxin-3 and alleviates Machado-Joseph disease.

              Machado-Joseph disease, also known as spinocerebellar ataxia type 3, is the most common of the dominantly inherited ataxias worldwide and is characterized by mutant ataxin-3 misfolding, intracellular accumulation of aggregates and neuronal degeneration. Here we investigated the implication of autophagy, the major pathway for organelle and protein turnover, in the accumulation of mutant ataxin-3 aggregates and neurodegeneration found in Machado-Joseph disease and we assessed whether specific stimulation of this pathway could mitigate the disease. Using tissue from patients with Machado-Joseph disease, transgenic mice and a lentiviral-based rat model, we found an abnormal expression of endogenous autophagic markers, accumulation of autophagosomes and decreased levels of beclin-1, a crucial protein in the early nucleation step of autophagy. Lentiviral vector-mediated overexpression of beclin-1 led to stimulation of autophagic flux, mutant ataxin-3 clearance and overall neuroprotective effects in neuronal cultures and in a lentiviral-based rat model of Machado-Joseph disease. These data demonstrate that autophagy is a key degradation pathway, with beclin-1 playing a significant role in alleviating Machado-Joseph disease pathogenesis.

                Author and article information

                Role: Editor
                PLoS One
                PLoS ONE
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                21 August 2014
                : 9
                : 8
                [1 ]CNC - Center for Neuroscience & 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 ]Lausanne University Hospital, Department of Clinical Neurosciences, Laboratory of Cellular and Molecular Neurotherapies, Lausanne, Switzerland
                Northwestern University, United States of America
                Author notes

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

                Conceived and designed the experiments: CN INF LPA. Performed the experiments: CN INF IO DA. Wrote the paper: CN INF LPA ND. Read and approved the manuscript: CN INF DA IO ND LPA.


                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: 14
                We thank the Portuguese Foundation for Science and Technology and FEDER funds by Programa Operacional Factores de Competitividade – COMPETE (PTDC/SAU-NEU/099307/2008, PTDC/SAU-FAR/116535/2010 and PEst-C/SAU/LA0001/2013-2014), the Richard Chin and Lily Lock Machado-Joseph Research Fund, the National Ataxia Foundation, and the Association Française pour les Myopathies (SB/NF/2010/2008 Number 15079CA) for funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                Biology and life sciences
                Nucleic Acids
                Cell Biology
                Molecular Cell Biology
                RNA interference
                Gene Expression
                Genetics of Disease
                Human Genetics
                Molecular Biology
                Molecular Biology Techniques
                Gene Therapy
                Medicine and Health Sciences
                Neurodegenerative Diseases
                Movement Disorders
                Research and Analysis Methods
                Animal Studies
                Animal Models of Disease
                Model Organisms
                Animal Models
                Mouse Models
                Research Design
                Clinical Research Design



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