. Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis. Ann Neurol. 2015 Oct;78(4):568-83. Epub 2015 Aug 29 PubMed.


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  1. In the years since the discovery that the hexanucleotide repeat expansion mutation in C9ORF72 is one of the major causes of ALS and FTLD, we have been presented with three major pathogenesis hypotheses: haploinsufficiency, RNA toxicity, and dipeptide repeat toxicity. Most of the subsequent research has focused on the last two, as they are thought to be more likely root causes of disease. Besides giving insight into disease pathogenesis, this research has made significant contributions to our understanding novel concepts in basic cellular mechanisms, such as RNA aggregation and repeat-associated non-ATG translation.

    In this paper, Janice Robertson’s group shifts the attention back to the C9ORF72 protein. They developed two antibodies that distinguish between the long and short isoforms of C9ORF72. The primary characterization of these antibodies reveals some striking ALS-related changes in their subcellular localization as well as possible interactions with two components of nucleocytoplasmic shuttling, a small GTPase RAN (ras-related nuclear protein) and KPNB1 (Importin-b1). The final figure in the paper, where the authors show correlation between RAN/KPNB1, C9ORF72 short form, and TDP-43 aggregation, is fascinating and, if proven by a larger study, will give significant insights into the involvement of nuclear transport in ALS. Importantly, it should also be noted that both RAN and KPNB1 are involved in cell cycle progression so it will be interesting to see if and where the C9ORF72 protein isoforms fit in that process. Five years ago we showed that TDP-43 is transported into the nucleus via the KPNB1 pathway (Nishimura et al., 2010) and recently we showed that loss of TDP-43 also leads to loss of components of nucleocytoplasmic shuttling in human cells, suggesting a feedback regulation of TDP-43 shuttling (Stalekar et al., 2015). One such component is RANBP1 (RAN binding protein 1), which is necessary for facilitating the GTPase function of RAN. Overall, these antibodies present an important new tool, which surely will enable significant advances in characterization of C9ORF72 function as well as understanding its role in disease.


    . Nuclear import impairment causes cytoplasmic trans-activation response DNA-binding protein accumulation and is associated with frontotemporal lobar degeneration. Brain. 2010 Jun;133(Pt 6):1763-71. PubMed.

    . Proteomic analyses reveal that loss of TDP-43 affects RNA processing and intracellular transport. Neuroscience. 2015 May 7;293:157-70. Epub 2015 Mar 2 PubMed.

    View all comments by Boris Rogelj
  2. Studies examining the role of the C9ORF72 protein isoforms have been hampered by the lack of commercial C9ORF72 antibodies with high specificity and sensitivity. Currently available reagents are also unable to individually detect the two C9ORF72 isoforms. This study describes the generation and preliminary use of novel isoform-specific C9ORF72 antibodies. The authors developed novel rabbit polyclonal antibodies against synthetic peptides specific to C9ORF72 long and short isoforms. They showed that the antibodies could selectively detect recombinant C9ORF72 isoforms. The antibodies also appear to show increased sensitivity for detection of C9ORF72 isoforms in immunoblotting studies of postmortem tissue samples compared to a Santa Cruz commercial antibody.

    Interestingly, the authors show different subcellular localization of the two C9ORF72 isoforms using immunohistochemistry in postmortem tissue, with the short isoform localizing to the nuclear envelope whereas the long isoform shows a diffuse cytoplasmic localization. Furthermore, the levels and subcellular localization of C9ORF72 isoforms are also differentially altered in ALS cases with and without the pathological C9ORF72 hexanucleotide expansion. The authors show that loss of nuclear envelope localization of C9ORF72 short form may correlate with TDP-43 pathology in spinal motor neurons of ALS cases. The study suggests that C9ORF72 could function in nucleocytoplasmic shuttling and that dysfunction of this pathway could be associated with TDP-43 pathology.

    They provide evidence that C9ORF72 may interact with the nucleocytoplasmic transport complex components importin-β1 and Ran-GTPase. Mislocalization of these proteins and C9ORF72 short isoform may correlate with TDP-43 pathology in ALS cases with or without pathological C9ORF72 repeat expansions. Evidence of a direct interaction between stably overexpressed C9ORF72 and importin complex components was shown. Further validation of this interaction by immunoprecipitation of endogenous C9ORF72 is required.

    The authors use recombinant C9ORF72 isoform expression and the addition of an antibody-blocking peptide for the purposes of showing antibody specificity for immunoblotting and immunohistochemistry. However, further studies should be performed using C9ORF72-deficient cells or tissue to verify specificity of the antibodies.

    This study emphasises how the hexanucleotide repeat expansion in C9ORF72 exerts multiple effects in the central nervous system. In addition to the generation of hexanucleotide-derived toxic RNA and dipeptide species there are differential effects on the protein levels and localization of C9ORF72 isoforms. Increasing evidence from animal and cellular models supports gain of function as the main pathological mechanism, whereas haploinsufficiency of C9ORF72 either represents epi-phenomena or could be a disease modifier. Mislocalization of C9ORF72 short form in motor neurons may also be a pathological correlate of ALS with TDP-43 pathology. The finding of C9ORF72 mislocalization in motor neurons from ALS cases without a hexanucleotide expansion and normal localisation in cerebellar Purkinje cells (that have been shown to contain RNA foci and C9RANT pathology) suggests this is not a specific consequence of the hexanucleotide expansion products. Further immunohistochemical studies of tissue with a higher burden of RNA foci and C9RANT pathology, such as frontal cortex and hippocampal tissue, may help address this issue.

    Further studies will be required to examine the role of C9ORF72 in nucleocytoplasmic shuttling and to explore whether mislocalization of C9ORF72 short form is a modifier of TDP-43 pathology in ALS or a downstream consequence of TDP-43 dysfunction. Bioinformatic analyses and preliminary functional studies of C9ORF72 suggest that it may be a member of the DENN family of GTP exchange factors (GEFs) and could regulate GTPase activity. Therefore C9ORF72 could regulate nucleocytoplasmic shuttling via GEF activity towards Ran-GTPase. However, a recent study published by Jeroen Pasterkamp and colleagues generated mice with neural-specific ablation C9ORF72 and showed no motor neuron degeneration or pathological hallmarks of ALS such as mislocalization of TDP-43 (Koppers et al., 2015). This would suggest that C9ORF72 deficiency does not affect localization of TDP-43 and that the reported mislocalization of C9ORF72 short form could be a result of dysfunction of TDP-43 or nucleocytoplasmic shuttling. Alterations in nucleocytoplasmic transport could also be a downstream effect of arginine-rich C9RANT products on Ran-GTPase alternative splicing (Kwon et al., 2014).


    . C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits. Ann Neurol. 2015 Sep;78(3):426-38. Epub 2015 Jul 3 PubMed.

    . Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science. 2014 Sep 5;345(6201):1139-45. Epub 2014 Jul 31 PubMed.

    View all comments by Adrian Waite

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  1. New C9ORF72 Antibodies Find Isoforms in Different Cellular Locations