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

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.

C9orf72 intermediate repeats are associated with corticobasal degeneration, increased C9orf72 expression and disruption of autophagy

“…This mechanism is also supported by publicly available SNP eQTL data, which shows that numerous SNPs on the expansion haplotype are associated with increased C9orf72 expression. This is in stark contrast to full C9orf72 expansion carriers, which have reduced C9orf72 mRNA and protein levels 2,7,42,55 . A similar observation has been made in FXTAS, where intermediate expansions increase FMR1 mRNA levels [56][57][58][59] , but large expansions lead to epigenetic silencing of the gene 60 .…”

Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders

“…Overexpression of DPR proteins using artificial ATG codons for their translation initiation leads to neurodegeneration in cell and animal models, notably through alteration of mitochondria (Dafinca et al , ; Lopez‐Gonzalez et al , ; Choi et al , ), DNA repair (Walker et al , ; Lopez‐Gonzalez et al , ), nuclear and nucleolar organization (White et al , ; Zhang et al , ), and/or nucleocytoplasmic transport (Kwon et al , ; May et al , ; Mizielinska et al , ; Wen et al , ; Zhang et al , , ; Freibaum et al , ; Jovičić et al , ; Tao et al , ; Boeynaems et al , ; Khosravi et al , ). Third, expanded G4C2 repeats promote DNA epigenetic changes that lead to decreased expression of C9ORF72 mRNA and protein levels in C9‐ALS/FTD individuals (DeJesus‐Hernandez et al , ; Gijselinck et al , ; Almeida et al , ; Waite et al , ; van Blitterswijk et al , ; Xiao et al , ; Saberi et al , ; Frick et al , ; Viodé et al , ). Interestingly, knockdown of C9ORF72 expression in zebrafish or in human motor neuron differentiated from iPS cells leads to some neuronal cell dysfunctions and cell death (Ciura et al , ; Shi et al , ).…”

PQLC2 recruits the C9orf72 complex to lysosomes in response to cationic amino acid starvation

“…A hexanucleotide repeat expansion in a noncoding region of the C9orf72 gene causes familial forms of amyotrophic lateral sclerosis and frontotemporal dementia (DeJesus-Hernandez et al, 2011; Gijselinck et al, 2012; Renton et al, 2011). Although the repeat expansion results in a reduction in C9orf72 mRNA and protein levels, the extent to which this is relevant for disease pathogenesis remains unclear (Belzil et al, 2013; DeJesus-Hernandez et al, 2011; Gijselinck et al, 2012; Shi et al, 2018; Viodé et al, 2018; Waite et al, 2014; Xi et al, 2013). Nonetheless, investigation of this topic has established that the C9orf72 protein is required for normal lysosome homeostasis in a variety of model systems, including mice, Caenorhabditis elegans , and cultured human cells (Amick et al, 2016; Corrionero and Horvitz, 2018; McAlpine et al, 2018; O’Rourke et al, 2016; Sullivan et al, 2016; Zhang et al, 2018).…”