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.

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    • "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). "
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    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.
    Journal of Cell Science 03/2015; 128(9). DOI:10.1242/jcs.165332 · 5.43 Impact Factor
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    • "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). "
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    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.
    Neurobiology of aging 01/2014; 35(7). DOI:10.1016/j.neurobiolaging.2014.01.016 · 5.01 Impact Factor
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    • "Most ALS cases appear in sporadic (sALS) forms; only about 10–15% have familial (fALS) history, and are clinically indistinguishable from sALS. fALSs have been associated with mutations in different genes, such as the superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma protein (FUS/TLS), optineurin (Robberecht and Philips, 2013), or the C9ORF72 gene (Dejesus-Hernandez et al., 2011; Renton et al., 2011; Ash et al., 2013; Lashley et al., 2013; Mori et al., 2013). Notably, also the wild type (wt) forms of the mutated fALS proteins may show aberrant behavior in sALS (e.g., oxydized wtSOD1, cleaved C-terminus of wtTDP-43, etc.) (Neumann et al., 2006; Daoud et al., 2009; Bosco and Landers, 2010; Bosco et al., 2010), suggesting the existence of a common pathological mechanism. "
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    ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a motoneuron disease characterized by misfolded proteins aggregation in affected motoneurons. In mutant SOD1 (mutSOD1) ALS models, aggregation correlates to impaired functions of proteasome and/or autophagy, both essential for the intracellular chaperone-mediated protein quality control (PQC), and a reduced mutSOD1 clearance from motoneurons. Skeletal muscle cells are also sensitive to mutSOD1 toxicity, but no mutSOD1 aggregates are formed in these cells, that might better manage mutSOD1 than motoneurons. Thus, we analysed in spinal cord and in muscle of transgenic (tg) G93A-SOD1 at presymptomatic (PS, 8 weeks) and symptomatic (S, 16 weeks) stages, and in age-matched control mice, whether mutSOD1 differentially modulates relevant PQC players, such as HSPB8, BAG3, and BAG1. Possible sex differences were also considered. No changes of HSPB8, BAG3 and BAG1 at PS stage (8 weeks) were seen in all tissues examined in tg G93A-SOD1 and control mice. At S stage (16 weeks), HSPB8 dramatically increased in skeletal muscle of tg G93A-SOD1 mice, while a minor increase occurred in spinal cord of male, but not female tg G93A-SOD1 mice. BAG3 expression increased both in muscle and spinal cord of tg G93A-SOD1 mice at S stage, BAG1 expression increased only in muscle of the same mice. Since, HSPB8-BAG3 complex assists mutSOD1 autophagic removal, we analysed two well-known autophagic markers, LC3 and p62. Both LC3 and p62 mRNAs were significantly up-regulated in skeletal muscle of tg G93A-SOD1 mice at S stage (16 weeks). This suggests that mutSOD1 expression induces a robust autophagic response specifically in muscle. Together these results demonstrate that, in muscle mutSOD1-induced autophagic response is much higher than in spinal cords. In addition, if mutSOD1 exerts toxicity in muscle, this may not be mediated by misfolded protein accumulation. It remains unclear whether in muscle mutSOD1 toxicity is related to aberrant autophagy activation.
    Frontiers in Cellular Neuroscience 11/2013; 7:234. DOI:10.3389/fncel.2013.00234 · 4.29 Impact Factor
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