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 b1 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 H exanucleotide (G 4 C 2 ) repeat expansions within a noncoding region of C9orf72 are the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), accounting for up to 50% of ALS, 29% of FTLD, and 88% of ALS/ FTLD patients, including familial and sporadic cases.

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

“…Protocols were evaluated based on total protein extraction and signal from C9ORF72 peptides. The low salt protocol without detergent resulted in low protein extraction yield and no signal for C9ORF72 peptides, although Western blot signal was previously reported for the short isoform (Xiao et al, 2015). Signals were observed for the three quantitative peptides selected for quantification (peptides 1–3) when applying the Triton containing mixtures, the latter showing higher intensity than that containing urea ( Figure 4A ).…”

“…Protein extraction protocols with tissue homogenization by mechanical shear recently published for C9ORF72 (Xiao et al, 2015), containing either low/high-salt content (i.e., 150 mM NaCl or 750 mM NaCl), 8 M urea or Triton X-100, were individually evaluated on tissues samples from frontal cortex ( Figure 4 ). These protocols were adapted to single use bead-beating Precellys tubes to parallelize protein extraction and avoid contamination between samples.…”

C9orf72 expansion differentially affects males with spinal onset amyotrophic lateral sclerosis

“…These include haploinsufficiency, toxic RNA interfering with the function of RNA-binding proteins or other cellular factors, and the presence of toxic dipeptide repeat proteins through Repeat-associated non-ATG (RAN) translation 25 26. Recent work has also pointed towards C9orf72 -induced pathology of nucleocytoplasmic transport processes 26–29.…”

Genetics insight into the amyotrophic lateral sclerosis/frontotemporal dementia spectrum

“…Both C9orf72 mRNA and the C9orf72 protein are highly expressed in the brain in mice and humans 38. Studies have reported lower levels of C9orf72 mRNA in patients carrying the expansions and reductions in the long C9orf72 isoform 39. However, the short isoform seems to accumulate at higher levels and relocalise to the plasma membrane, suggesting a potential mechanism of toxicity, although the significance of these findings is unclear.…”