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      Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion

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          Abstract

          An intronic G 4C 2 hexanucleotide repeat expansion in C9ORF72 is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several mechanisms including RNA toxicity, repeat-associated non-AUG translation mediated dipeptide protein aggregates, and haploinsufficiency of C9orf72 have been implicated in the molecular pathogenesis of this disorder. The aims of this study were to compare the use of two different Southern blot probes for detection of repeat expansions in an amyotrophic lateral sclerosis and frontotemporal lobar degeneration pathological cohort and to determine the levels of C9orf72 transcript variants and protein isoforms in patients versus control subjects. Our Southern blot studies identified smaller repeat expansions (250–1800 bp) that were only detectable with the flanking probe highlighting the potential for divergent results using different Southern blotting protocols that could complicate genotype–phenotype correlation studies. Further, we characterize a new C9orf72 antibody and show for the first time decreased C9orf72 protein levels in the frontal cortex from patients with a pathological hexanucleotide repeat expansion. These data suggest that a reduction in C9orf72 protein may be a consequence of the disease.

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          RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.

          A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9.

            C Liquori (2001)
            Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19q13 (DM1) or 3q21 (DM2/PROMM). DM1 is caused by a CTG expansion in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). Several mechanisms have been invoked to explain how this mutation, which does not alter the protein-coding portion of a gene, causes the specific constellation of clinical features characteristic of DM. We now report that DM2 is caused by a CCTG expansion (mean approximately 5000 repeats) located in intron 1 of the zinc finger protein 9 (ZNF9) gene. Parallels between these mutations indicate that microsatellite expansions in RNA can be pathogenic and cause the multisystemic features of DM1 and DM2.
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              Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease

              The tripartite DENN module, comprised of a N-terminal longin domain, followed by DENN, and d-DENN domains, is a GDP-GTP exchange factor (GEFs) for Rab GTPases, which are regulators of practically all membrane trafficking events in eukaryotes. Using sequence and structure analysis we identify multiple novel homologs of the DENN module, many of which can be traced back to the ancestral eukaryote. These findings provide unexpected leads regarding key cellular processes such as autophagy, vesicle-vacuole interactions, chromosome segregation, and human disease. Of these, SMCR8, the folliculin interacting protein-1 and 2 (FNIP1 and FNIP2), nitrogen permease regulator 2 (NPR2), and NPR3 are proposed to function in recruiting Rab GTPases during different steps of autophagy, fusion of autophagosomes with the vacuole and regulation of cellular metabolism. Another novel DENN protein identified in this study is C9ORF72; expansions of the hexanucleotide GGGGCC in its first intron have been recently implicated in amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). While this mutation is proposed to cause a RNA-level defect, the identification of C9ORF72 as a potential DENN-type GEF raises the possibility that at least part of the pathology might relate to a specific Rab-dependent vesicular trafficking process, as has been observed in the case of some other neurological conditions with similar phenotypes. We present evidence that the longin domain, such as those found in the DENN module, are likely to have been ultimately derived from the related domains found in prokaryotic GTPase-activating proteins of MglA-like GTPases. Thus, the origin of the longin domains from this ancient GTPase-interacting domain, concomitant with the radiation of GTPases, especially of the Rab clade, played an important role in the dynamics of eukaryotic intracellular membrane systems.
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                Author and article information

                Contributors
                Journal
                Neurobiol Aging
                Neurobiol. Aging
                Neurobiology of Aging
                Elsevier
                0197-4580
                1558-1497
                1 July 2014
                July 2014
                : 35
                : 7
                : 1779.e5-1779.e13
                Affiliations
                [a ]Institute of Psychological Medicine and Clinical Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics, School of Medicine, Cardiff University, Cathays, Cardiff, UK
                [b ]Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford, UK
                [c ]Department of Pathology, School of Medicine, Cardiff University, Cardiff, UK
                [d ]Department of Clinical Neuroscience, UCL Institute of Neurology, Royal Free Hospital, London, UK
                Author notes
                []Corresponding author at: Institute of Psychological Medicine and Clinical Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics, School of Medicine, Cardiff University, Hadyn Ellis Building, Maindy Road, Cathays, Cardiff, CF24 4HQ, UK. Tel.: +44 (0)2920 688460; fax: +44 (0)2920 687068. waitea@ 123456cf.ac.uk
                Article
                S0197-4580(14)00017-7
                10.1016/j.neurobiolaging.2014.01.016
                3988882
                24559645
                dbbabcce-356b-4cf5-bf19-3e25b4fd1641
                © 2014 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

                History
                : 20 September 2013
                : 9 December 2013
                : 12 January 2014
                Categories
                Genetic Report Abstract

                Neurosciences
                amyotrophic lateral sclerosis,c9orf72,frontotemporal dementia,frontotemporal lobar degeneration,repeat expansion,southern blotting

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