Subcellular Localizations of C9orf72 in Amyotrophic Lateral Sclerosis
Reporter: Aviva Lev-Ari, PhD, RN
Isoform Specific Antibodies Reveal Distinct Subcellular Localizations of C9orf72 in Amyotrophic Lateral Sclerosis.
Xiao S1, MacNair L1,2, McGoldrick P1, McKeever PM1,2, McLean JR1, Zhang M1, Keith J2,3, Zinman L2,3, Rogaeva E1, Robertson J1.
Abstract
OBJECTIVE:
A noncoding hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). It has been reported that the repeat expansion causes a downregulation of C9orf72 transcripts, suggesting that haploinsufficiency may contribute to disease pathogenesis. Two protein isoforms are generated from three alternatively spliced transcripts of C9orf72; a long form (C9-L) and a short form (C9-S) and their function(s) are largely unknown due to lack of specific antibodies.
METHODS:
To investigate C9orf72 protein properties, we developed novel antibodies that recognize either C9-L or C9-S. Multiple techniques including western blot, immunohistochemistry and co-immunoprecipitation were used to determine the expression levels and subcellular localizations of C9-L and C9-S.
RESULTS:
Investigation of expression of C9-L and C9-S demonstrated distinct biochemical profiles, region-specific changes and distinct subcellular localizations in ALS tissues. In particular, C9-L antibody exhibited a diffuse cytoplasmic staining in neurons, and labeled large speckles in cerebellar Purkinje cells. In contrast, C9-S antibody gave very specific labeling of the nuclear membrane in healthy neurons, with apparent re-localization to the plasma membrane of diseased motor neurons in ALS. Co-immunoprecipitation experiments revealed an interaction of the C9-isoforms with both Importin β1 and Ran-GTPase, components of the nuclear pore complex.
INTERPRETATION:
Using these antibodies, we have shown that C9orf72 may be involved in nucleocytoplasmic shuttling and this may have relevance to pathophysiology of ALS/FTLD. Our antibodies have provided improved detection of C9orf72 protein isoforms, which will help elucidate its physiological function and role in ALS/FTLD. This article is protected by copyright. All rights reserved.
SOURCE
http://www.ncbi.nlm.nih.gov/pubmed/26174152
Journal Reference:
- Shangxi Xiao, Laura MacNair, Philip McGoldrick, Paul M McKeever, Jesse R McLean, Ming Zhang, Julia Keith, Lorne Zinman, Ekaterina Rogaeva, Janice Robertson. Isoform Specific Antibodies Reveal Distinct Subcellular Localizations of C9orf72 in Amyotrophic Lateral Sclerosis.Annals of Neurology, 2015; DOI: 10.1002/ana.24469
University of Toronto research team has discovered new details about a key gene involved in ALS, perhaps humanity’s most puzzling, intractable disease.
In this fatal disorder with no effective treatment options, scientists (including members of U of T) achieved a major breakthrough in 2011 when they discovered mutations in the gene C9orf72, as the most frequent genetic cause of ALS and frontotemporal dementia. But little was known about how this gene and its related protein worked in the cell.
To solve this problem, Professor Janice Robertson and her team at the Tanz Centre for Research in Neurodegenerative Diseases developed novel antibodies that not only specifically detected C9orf72 in human tissues, but could also distinguish between both the long and short isoforms.
“Using these antibodies we have made the remarkable discovery that C9orf72 is localized to the nuclear membrane in healthy neurons, but is mislocalized to the plasma (outer membrane) in diseased neurons,” says Robertson, whose research was published July 14 online in the journal Annals of Neurology.
Robertson and her team also showed that C9orf72 directly interacts with components of the nuclear shuttling complex, which is responsible for the movement of proteins across the nuclear membrane. One such protein is TDP-43, which normally resides in the nucleus but is wrongly localized to the cytoplasm in diseased neurons in ALS. TDP-43 accumulation and aggregation in the cytoplasm diagnoses most ALS cases — but the link with C9orf72 was absent.
Now through the use of the C9orf72 antibodies the Robertson lab has found that loss of C9orf72 from the nuclear membrane correlates with TDP-43 pathology. These results suggest that defects in C9orf72 affect the proper functioning of the nuclear shuttling complex, resulting in TDP-43 build up in the cytoplasm.
“We’ve discovered a link between the genetic cause of ALS and its pathology that appears to be important for all cases, not just familial ones,” says Robertson, a Canada Research Chair in ALS. “The possible involvement of C9orf72 in the shuttling between nucleus and cytoplasm opens intriguing new avenues of research into the causes of ALS — and hopefully, one day an effective treatment or cure.”
SOURCE
http://www.sciencedaily.com/releases/2015/07/150715122405.htm
Journal Reference:
- Shangxi Xiao, Laura MacNair, Philip McGoldrick, Paul M McKeever, Jesse R McLean, Ming Zhang, Julia Keith, Lorne Zinman, Ekaterina Rogaeva, Janice Robertson. Isoform Specific Antibodies Reveal Distinct Subcellular Localizations of C9orf72 in Amyotrophic Lateral Sclerosis.Annals of Neurology, 2015; DOI: 10.1002/ana.24469
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