Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis.

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 owing 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 coimmunoprecipitation, 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 relocalization to the plasma membrane of diseased motor neurons in ALS. Coimmunoprecipitation 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. Ann Neurol 2015;78:568–583