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      ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice

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          Abstract

          Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG) 31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissue-specific effects, which may be partially alleviated by the induction of FBXW8.

          Author Summary

          Frequent age-associated neurodegenerative disorders like Alzheimer's, Parkinson's, and Lou Gehrig's disease are being elucidated molecularly by studying rare heritable variants. Various hereditary neurodegenerative disorders are caused by polyglutamine expansions in different proteins. In spite of this common pathogenesis and the pathological aggregation of most affected proteins, investigators were puzzled that the pattern of affected neuron population varies and that molecular mechanisms seem different between such disorders. The polyglutamine expansions in the Ataxin-2 (ATXN2) protein are exceptional in view of the lack of aggregate clumps in nuclei of affected Purkinje neurons and well documented alterations of RNA processing in the resulting disorders SCA2 and ALS. Here, as a faithful disease model and to overcome the unavailability of autopsied patient brain tissues, we generated and characterized an ATXN2-CAG42-knock-in mouse mutant. Our data show that the unspecific, chronically present mutation leads to progressive insolubility and to reduced soluble levels of the disease protein and of an interactor protein, which modulates RNA processing. Compensatory efforts are particularly weak in vulnerable tissue. They appear to include the increased degradation of the toxic disease protein by FBXW8. Thus the link between protein and RNA pathology becomes clear, and crucial molecular targets for preventive therapy are identified.

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          Most cited references61

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          The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain

          The Rbfox family of RNA binding proteins regulates alternative splicing of many important neuronal transcripts but their role in neuronal physiology is not clear 1 . We show here that central nervous system (CNS)-specific deletion of the Rbfox1 gene results in heightened susceptibility to spontaneous and kainic acid-induced seizures. Electrophysiological recording reveals a corresponding increase in neuronal excitability in the dentate gyrus of the knockout mice. Whole transcriptome analyses identify multiple splicing changes in the Rbfox1−/− brain with few changes in overall transcript abundance. These splicing changes alter proteins that mediate synaptic transmission and membrane excitation, some of which are implicated in human epilepsy. Thus, Rbfox1 directs a genetic program required in the prevention of neuronal hyperexcitation and seizures. The Rbfox1 knockout mice provide a new model to study the post-transcriptional regulation of synaptic function.
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            Cloning of the gene for spinocerebellar ataxia 2 reveals a locus with high sensitivity to expanded CAG/glutamine repeats.

            Two forms of the neurodegenerative disorder spinocerebellar ataxia are known to be caused by the expansion of a CAG (polyglutamine) trinucleotide repeat. By screening cDNA expression libraries, using an antibody specific for polyglutamine repeats, we identified six novel genes containing CAG stretches. One of them is mutated in patients with spinocerebellar ataxia linked to chromosome 12q (SCA2). This gene shows ubiquitous expression and encodes a protein of unknown function. Normal SCA2 alleles (17 to 29 CAG repeats) contain one to three CAAs in the repeat. Mutated alleles (37 to 50 repeats) appear particularly unstable, upon both paternal and maternal transmissions. The sequence of three of them revealed pure CAG stretches. The steep inverse correlation between age of onset and CAG number suggests a higher sensitivity to polyglutamine length than in the other polyglutamine expansion diseases.
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              Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

              Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                1553-7390
                1553-7404
                August 2012
                August 2012
                30 August 2012
                : 8
                : 8
                : e1002920
                Affiliations
                [1 ]Experimental Neurology, Department of Neurology, Goethe University Medical School, Frankfurt am Main, Germany
                [2 ]Department of Clinical Neuroanatomy, Dr. Senckenbergisches Chronomedizinisches Institut, Goethe University Medical School, Frankfurt am Main, Germany
                [3 ]Institute of Medical Genetics, Eberhard Karls University, Tübingen, Germany
                University of Minnesota, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: ED SG JN CS GA. Performed the experiments: ED MVH SG MA JN MW. Analyzed the data: ED MVH SG MA JN UR MW GA. Contributed reagents/materials/analysis tools: SG MA MW GA. Wrote the paper: ED GA.

                Article
                PGENETICS-D-12-00575
                10.1371/journal.pgen.1002920
                3431311
                22956915
                3bd30c80-c1df-4c5e-a0b3-fa76a904e206
                Copyright @ 2012

                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.

                History
                : 3 March 2012
                : 10 July 2012
                Page count
                Pages: 17
                Funding
                The project had financial support from the DFG (AU96/11-1, 13-1, 14-1), the European Union EUROSCA project, the Dr. Senckenbergische Stiftung in Frankfurt am Main, and the Stiftung für Hoffnung in Köln, Germany. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Genetics
                Gene Expression
                Protein Translation
                Heredity
                Genotypes
                Phenotypes
                Molecular Genetics
                Gene Regulation
                Animal Genetics
                Genetics of Disease
                Histology
                Model Organisms
                Animal Models
                Mouse
                Neuroscience
                Behavioral Neuroscience
                Neurobiology of Disease and Regeneration

                Genetics
                Genetics

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