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

ObjectiveA 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.
MethodsTo 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.
ResultsInvestigation 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‐GTPa...

Source: Annals of Neurology - August 29, 2015 Category: Neurology Authors: Shangxi Xiao, Laura MacNair, Philip McGoldrick, Paul M. McKeever, Jesse R. McLean, Ming Zhang, Julia Keith, Lorne Zinman, Ekaterina Rogaeva, Janice Robertson Tags: Research Article Source Type: research