New C9ORF72 Antibodies Find Isoforms in Different Cellular Locations

The C9ORF72 gene has been linked to both amyotrophic lateral sclerosis and frontotemporal dementia. Researchers trying to understand the function of the C9ORF72 protein have, until recently, had to make do with fairly poor antibodies. These were unable to distinguish the two natural 54- and 24-kilodalton isoforms, which arise through alternative splicing. In the July 14 Annals of Neurology online, scientists report two new C9ORF72 antibodies. Developed by Shangxi Xiao in the University of Toronto laboratory of Janice Robertson, they distinguish between the short and long isoforms, allowing the scientists to analyze these two versions separately. The antibodies revealed that the short form populates the nuclear membrane of neurons, while its long counterpart remains in the cytoplasm. Further, it looks like the short version might usher another ALS- and FTD-linked protein into the nucleus—TDP-43.

Multiple membranes.

In spinal motor neurons from neurologically healthy people (top), the short form of C9ORF72 attaches to the nuclear membrane, but it localizes to the plasma membrane in tissue from people with ALS (bottom). [Image courtesy of Wiley/Annals of Neurology ” American Neurological Association]

While hexanucleotide expansions in C9ORF72 first attracted scientists to this protein, they still have no clear idea what the wild-type protein does (see Sep 2011 news). Commercial antibodies to C9ORF72 were already available in 2011, but they offered poor specificity and sensitivity, said Adrian Waite of the Cardiff University School of Medicine in the United Kingdom. Waite tackled the problem with his own C9ORF72 antibody, which recognizes the amino terminus of the protein. However, it does not distinguish between the two isoforms (Waite et al., 2014). Tania Gendron of the Mayo Clinic in Jacksonville, Florida, said this pan-C9ORF72 antibody is a useful tool, but that isoform-specific antibodies were long overdue. To deliver on those, Xiao took advantage of a terminal lysine unique to the short isoform to generate a synthetic peptide to match. He used this and a peptide found only in the long isoform to immunize rabbits and generate polyclonal antibodies. Now, Xiao and Robertson offer a first glimpse of how these antibodies can help scientists understand C9ORF72. “I think they are going to be a phenomenal resource,” Gendron told Alzforum.

On western blots of human cerebellar extracts, Xiao determined that the short form of C9ORF72 may form a dimer. The antibody to the short form recognized two proteins, one being twice the predicted size. The antibody to the large isoform detected a single band of the expected 54 kDa. Xiao found the same pattern in postmortem extracts from controls and from ALS cases—with or without the C9ORF72 expansion gene. “The antibodies look really clean,” Gendron commented.

To see where the two isoforms localize in cells, Xiao turned to immunohistochemistry. In spinal motor neurons from controls and ALS cases, the long C9ORF72 typically appeared in the cytoplasm, offering little clue to its function. In contrast, the short form settled on the nuclear membrane in the majority of control neurons. Interestingly, it was absent from the nucleus in about 60 percent of the neurons in ALS cases. It turned up on the plasma membrane instead (see image above). The pattern was the same whether the ALS was linked to C9ORF72 expansions or not.

Intrigued by the altered localization of the short isoform, Xiao investigated further. In controls, the short form co-localized on the nucleus with Ran-GTPase and importin-β1, which regulate the trafficking of proteins into the nucleus. Like short C9ORF72, these two nuclear import factors were missing from the nuclear membranes of many cells in ALS cases. To check if C9ORF72 directly interacted with these nuclear shuttlers, Xiao used mouse neuroblastoma N2a cultures. When he overexpressed C9ORF72 in those cells, it co-immunoprecipitated with both RAN-GTPase and importin-β1. Thus, the authors suspect C9ORF72 might participate in nuclear import as well.

Other studies have also linked nuclear shuttling to ALS. TDP-43 normally resides in the nucleus but is often found in the cytoplasm of diseased neurons. Scientists have blamed faulty nuclear uptake for this mislocalization (Nishimura et al., 2010; Ward et al., 2014). Might C9ORF72 be involved? Xiao stained human spinal cords for TDP-43 and short C9ORF72. In cells where C9ORF72 had quit the nuclear membrane, TDP-43 was mislocalized to the cytoplasm.

Based on these data, Robertson and Xiao theorize that C9ORF72 abnormalities could cause the short isoform to depart the nuclear membrane, making it impossible for TDP-43 to get into the nucleus. Notably, because the short C9ORF72 vacated the nucleus in sporadic cases of ALS, the authors believe that not just the C9 hexanucleotide expansion but other factors influence C9ORF72 localization and thus TDP-43 import. They plan to look at this in more detail.

Gendron was intrigued by the proposed mechanism, but said she would like to see direct evidence that C9ORF72 participates in nuclear import, for example by knocking down the gene in human cell models.

Waite pointed out that bioinformatics analysis of C9ORF72 suggests it could be a GTP exchange factor, and he speculated it might be on the nuclear membrane to regulate Ran-GPTase (see Jan 2013 news). However, he noted other recent data that might contradict Xiao’s model (see full comment below). For example, one group eliminated neural C9ORF72 in mice and saw no TDP-43 mislocalization (see Jun 2015 news). Robertson pointed out that mice lack the short C9ORF72 isoform, and that their TDP-43 differs from the human version in both sequence and splice variants. She said C9ORF72 knockout mice would not necessarily mimic all aspects of ALS in people.

Reference:

Xiao S, MacNair L, McGoldrick P, McKeever PM, McLean JR, Zhang M, Keith J, Zinman L, Rogaeva E, Robertson J. Isoform Specific Antibodies Reveal Distinct Subcellular Localizations of C9orf72 in Amyotrophic Lateral Sclerosis. Ann Neurol. 2015 Oct;78(4):568-83. Epub 2015 Jul 14. [Pubmed].


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disease-als disease-ftd topic-newmethods topic-preclinical
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