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

Arthur Viodé,François Becher,Agnès Camuzat,Christophe Junot,Foudil Lamari,Neuroceb Brain Bank ,Morwena Latouche,Fanny Elahi,Vincent Anquetil,Isabelle Le Ber,François Fenaille,Clémence Fournier

doi:10.3389/fnins.2018.00589

Abstract

Frontotemporal dementia (FTD) is a fatal neurodegenerative disease characterized by behavioral and language disorders. The main genetic cause of FTD is an intronic hexanucleotide repeat expansion (G4C2)n in the C9ORF72 gene. A loss of function of the C9ORF72 protein associated with the allele-specific reduction of C9ORF72 expression is postulated to contribute to the disease pathogenesis. To better understand the contribution of the loss of function to the disease mechanism, we need to determine precisely the level of reduction in C9ORF72 long and short isoforms in brain tissue from patients with C9ORF72 mutations. In this study, we developed a sensitive and robust mass spectrometry (MS) method for quantifying C9ORF72 isoform levels in human brain tissue without requiring antibody or affinity reagent. An optimized workflow based on surfactant-aided protein extraction and pellet digestion was established for optimal recovery of the two isoforms in brain samples. Signature peptides, common or specific to the isoforms, were targeted in brain extracts by multiplex MS through the parallel reaction monitoring mode on a Quadrupole–Orbitrap high resolution mass spectrometer. The assay was successfully validated and subsequently applied to frontal cortex brain samples from a cohort of FTD patients with C9ORF72 mutations and neurologically normal controls without mutations. We showed that the C9ORF72 short isoform in the frontal cortices is below detection threshold in all tested individuals and the C9ORF72 long isoform is significantly decreased in C9ORF72 mutation carriers.

Highlights

Frontotemporal dementia (FTD) is the second most prevalent neurodegenerative disease before the age of 65, after Alzheimer disease
C9 open reading frame 72 (C9ORF72) isoforms have solely been investigated by Western blot which relies on the availability of antibodies whose specificity has to be characterized and validated (Liu et al, 2006; Davidson et al, 2018)
We found that Triton X-100 facilitated the extraction of the C9ORF72 proteins, in agreement with higher extraction yield reported with detergent based protocols in comparison to organic solvents or chaotropic reagents such as urea, in fatty tissue such as the brain (Shevchenko et al, 2012)

Summary

Introduction

Frontotemporal dementia (FTD) is the second most prevalent neurodegenerative disease before the age of 65, after Alzheimer disease. Amyotrophic lateral sclerosis (ALS) caused by motor neuron degeneration is associated with FTD in 15% of patients or families. Familial forms of FTD, accounting for about 20–50% of cases (Rosso, 2003) are mainly caused by mutations in three major genes: granulin (GRN), microtubule-associated protein tau (MAPT) and C9 open reading frame 72 (C9ORF72). The two protein isoforms have been shown by immunofluorescence to have distinct cellular localization with the C9-S lozalizing to the nuclear membrane and C9-L to the cytoplasm (Xiao et al, 2016). This observation suggests that the two isoforms have a different function, while the precise function of the protein has not been clearly determined. A potential role in endosomal transport and autophagy was reported through interaction with Rab-GTPases (Zhang et al, 2012; Levine et al, 2013)

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