HnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations

Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Schillerstr. 44, 80336, Munich, Germany.
Acta Neuropathologica (Impact Factor: 10.76). 02/2013; 125(3). DOI: 10.1007/s00401-013-1088-7
Source: PubMed


Genetic analysis revealed the hexanucleotide repeat expansion GGGGCC within the regulatory region of the gene C9orf72 as the most common cause of familial amyotrophic lateral sclerosis and the second most common cause of frontotemporal lobar degeneration. Since repeat expansions might cause RNA toxicity via sequestration of RNA-binding proteins, we searched for proteins capable of binding to GGGGCC repeats. In vitro-transcribed biotinylated RNA containing hexanucleotide GGGGCC or, as control, AAAACC repeats were incubated with nuclear protein extracts. Using stringent filtering protocols 20 RNA-binding proteins with a variety of different functions in RNA metabolism, translation and transport were identified. A subset of these proteins was further investigated by immunohistochemistry in human autopsy brains. This revealed that hnRNP A3 formed neuronal cytoplasmic and intranuclear inclusions in the hippocampus of patients with C9orf72 repeat extensions. Confocal microcopy showed that these inclusions belong to the group of the so far enigmatic p62-positive/TDP-43 negative inclusions characteristically seen in autopsy cases of diseased C9orf72 repeat expansion carriers. Thus, we have identified one protein component of these pathognomonic inclusions.

Download full-text


Available from: Ignasi Forne
    • "heterogeneous nuclear ribonucleoprotein (hnRNP) H1/F, hnRNP A1, and Aly/REF export factor (Cooper-Knock et al., 2014; Lee et al., 2013; Mori et al., 2013b). In a Drosophila model, the RNABP Pur a binds to RNA C9ORF72 expanded repeats causing neurodegeneration (Xu et al., 2013 ). "
    [Show abstract] [Hide abstract]
    ABSTRACT: An astonishing number of neurological diseases result from expansion of unstable repetitive sequences causing alterations in key neuronal processes. Some are progressive late-onset conditions related to aging, such as the spinocerebellar ataxias. In several of these pathologies, the expanded repeat is transcribed, producing an expanded RNA repeat that causes neurodegeneration by a complex mechanism, comprising 3 main pathways. These include (1) accumulation in the nucleus of RNA foci, resulting from sequestration of RNA-binding proteins functioning in important neuronal cascades; (2) decrease in availability of RNA-binding proteins, such as splicing factors, causing alternative splicing misregulation with imbalance in the expression ratio of neuronal isoforms; and (3) generation of neurotoxic peptides, produced from repeat-associated non-ATG-initiated translation across the RNA repeat, in all reading frames. Recently, 2 pathologies characterized by impaired motor function, cognitive decline, or/and degeneration of motor neurons have been found that have broaden our understanding of these diseases. Moreover, the finding of compromised nucleocytoplasmic transport opens new avenues for research. This review will cover the amazing progress regarding these conditions.
    No preview · Article · Dec 2015
    • "To get insights into this process, we have characterized the major binding partners of a (G4C2) 31 repeat. Our analysis showed that a substantial number of binding partners are RNA regulatory factors, as expected by the nature of the repeat sequence and in line with previous analyses showing that splicing and translation regulatory factors are binding partners of C9orf72 (Cooper- Knock et al., 2014; Donnelly et al., 2013; Haeusler et al., 2014; Mori et al., 2013; Xu et al., 2013). In our study, translational regulators are particularly represented: these include initiation and elongation factors (EF1a, eIF2a, eIF2b, eIF2c), but also Pura, Pur-beta and other translation regulatory proteins (ILF2, ILF3 and RAX). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A common feature of non-coding repeat expansion disorders is the accumulation of RNA repeats as RNA foci in the nucleus and/or cytoplasm of affected cells. These RNA foci can be toxic by sequestering RNA-binding proteins, thus affecting various steps of post-transcriptional gene regulation. However, the precise step that is affected by C9orf72 GGGGCC (G4C2) repeat expansion, the major genetic cause of Amyotrophic Lateral Sclerosis, is still poorly defined. In this work, we set out to characterise these mechanisms by identifying C9orf72 RNA binding proteins. Sequestration of some of these factors into RNA foci was observed when a (G4C2)31 repeat was expressed in NSC34 and HeLa cells. Most notably, (G4C2)31 repeats widely affected the distribution of Pur-alpha and its binding partner FMRP, which accumulate in intra-cytosolic granules that are positive for stress granules markers. Accordingly, translational repression is induced. Interestingly, this effect is associated to a marked accumulation of poly(A) mRNAs in cell nuclei. Thus, defective trafficking of mRNA, as a consequence of impaired nuclear mRNA export, might affect translation efficiency and contribute to the pathogenesis of C9orf72 ALS.
    No preview · Article · Mar 2015 · Journal of Cell Science
  • Source
    • "Dysfunction of vesicular trafficking processes such as endosomal-lysosomal trafficking and autophagy may be a pathological consequence of the C9ORF72 hexanucleotide repeat and has been implicated in other genetic forms of motor neuron degeneration and FTD (Devon et al., 2006; Filimonenko et al., 2007; Ju et al., 2009; Tumbarello et al., 2013; Wild et al., 2011). A collection of studies analyzing neurons differentiated from iPSCs derived from pathological expansion carriers point toward a gain-of-function mechanism resulting from toxic repeat-containing RNA inclusions and insoluble peptide aggregates generated by non-AUG mediated translation of the hexanucleotide repeat (Almeida et al., 2013; Ash et al., 2013; DeJesus-Hernandez et al., 2011; Donnelly et al., 2013; Lagier-Tourenne et al., 2013; Mori et al., 2013a, 2013b; Sareen et al., 2013). These potentially neurotoxic molecules are a result of bidirectional transcription of the expanded repeat (Gendron et al., 2013; Lagier-Tourenne et al., 2013; Mizielinska et al., 2013; Zu et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: An intronic G4C2 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.
    Full-text · Article · Jan 2014 · Neurobiology of aging
Show more