title: Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis.
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 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Shangxi
authors.initials: S
authors.lastname: Xiao
authors.affiliation: Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Laura
authors.initials: L
authors.lastname: MacNair
authors.affiliation: Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Philip
authors.initials: P
authors.lastname: McGoldrick
authors.affiliation: Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Paul M
authors.initials: PM
authors.lastname: McKeever
authors.affiliation: Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jesse R
authors.initials: JR
authors.lastname: McLean
authors.affiliation: Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.|Sunnybrook Health Sciences Center, Toronto, Ontario, Canada.
authors.forename: Julia
authors.initials: J
authors.lastname: Keith
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.|Sunnybrook Health Sciences Center, Toronto, Ontario, Canada.
authors.forename: Lorne
authors.initials: L
authors.lastname: Zinman
authors.affiliation: Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Rogaeva
authors.affiliation: Tanz Center for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Janice
authors.initials: J
authors.lastname: Robertson
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000690:Amyotrophic Lateral Sclerosis / Q000175:diagnosis* / Q000378:metabolism; D000906:Antibodies / Q000032:analysis* / Q000378:metabolism; D000073885:C9orf72 Protein; D002531:Cerebellum / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009046:Motor Neurons / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D020033:Protein Isoforms / Q000032:analysis / Q000096:biosynthesis; D011506:Proteins / Q000032:analysis* / Q000378:metabolism
chemical_list: D000906:Antibodies; D000073885:C9orf72 Protein; C568651:C9orf72 protein, human; D020033:Protein Isoforms; D011506:Proteins