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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.

SUBMITTER: Viode A

PROVIDER: S-EPMC6122177 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Viodé Arthur A Fournier Clémence C Camuzat Agnès A Fenaille François F Latouche Morwena M Elahi Fanny F Le Ber Isabelle I Junot Christophe C Lamari Foudil F Anquetil Vincent V Becher François F

Frontiers in neuroscience 20180828

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 dis ...[more]

PMID: 30210275

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Project description:An expanded GGGGCC hexanucleotide repeat in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration associated with TDP-43 pathology (FTLD-TDP). In addition to TDP-43-positive neuronal and glial inclusions, C9ORF72-linked FTLD-TDP has characteristic TDP-43-negative neuronal cytoplasmic and intranuclear inclusions as well as dystrophic neurites in the hippocampus and cerebellum. These lesions are immunopositive for ubiquitin and ubiquitin-binding proteins, such as sequestosome-1/p62 and ubiquilin-2. Studies examining the frequency of the C9ORF72 mutation in clinically probable Alzheimer's disease (AD) have found a small proportion of AD cases with the mutation. This prompted us to systematically explore the frequency of Alzheimer-type pathology in a series of 17 FTLD-TDP cases with mutations in C9ORF72 (FTLD-C9ORF72). We identified four cases with sufficient Alzheimer-type pathology to meet criteria for intermediate-to-high-likelihood AD. We compared AD pathology in the 17 FTLD-C9ORF72 to 13 cases of FTLD-TDP linked to mutations in the gene for progranulin (FTLD-GRN) and 36 cases of sporadic FTLD (sFTLD). FTLD-C9ORF72 cases had higher Braak neurofibrillary tangle stage than FTLD-GRN. Increased tau pathology in FTLD-C9ORF72 was assessed with thioflavin-S fluorescent microscopy-based neurofibrillary tangle counts and with image analysis of tau burden in temporal cortex and hippocampus. FTLD-C9ORF72 had significantly more neurofibrillary tangles and higher tau burden compared with FTLD-GRN. The differences were most marked in limbic regions. On the other hand, sFTLD and FTLD-C9ORF72 had a similar burden of tau pathology. These results suggest FTLD-C9ORF72 has increased propensity for tau pathology compared to FTLD-GRN, but not sFTLD. The accumulation of tau as well as lesions immunoreactive for ubiquitin and ubiquitin-binding proteins (p62 and ubiquilin-2) suggests that mutations in C9ORF72 may involve disrupted protein degradation that favors accumulation of multiple different proteins.

Project description:Diminished basal parasympathetic nervous system activity is a feature of frontotemporal dementia that relates to left frontoinsula dysfunction and empathy impairment. Individuals with a pathogenic expansion of the hexanucleotide repeat in chromosome 9 open reading frame 72 (C9orf72), the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, provide a unique opportunity to examine whether parasympathetic activity is disrupted in genetic forms of frontotemporal dementia and to investigate when parasympathetic deficits manifest in the pathophysiological cascade. We measured baseline respiratory sinus arrhythmia, a parasympathetic measure of heart rate variability, over two minutes in a sample of 102 participants that included 19 asymptomatic expansion carriers (C9+ asymp), 14 expansion carriers with mild cognitive impairment (C9+ MCI), 16 symptomatic expansion carriers with frontotemporal dementia (C9+ FTD), and 53 expansion-negative healthy controls (C9- HC) who also underwent structural magnetic resonance imaging. In follow-up analyses, we compared baseline respiratory sinus arrhythmia in the C9+ FTD group with an independent age-, sex-, and clinical severity-matched group of 26 people with sporadic behavioral variant frontotemporal dementia. The Frontotemporal Lobar Degeneration-modified Clinical Dementia Rating-Sum of Boxes score was used to quantify behavioral symptom severity, and informant ratings on the Interpersonal Reactivity Index provided measures of participants' current emotional (empathic concern) and cognitive (perspective-taking) empathy. Results indicated that the C9+ FTD group had lower baseline respiratory sinus arrhythmia than the C9+ MCI, C9+ asymp, and C9- HC groups, a deficit that was comparable to that of sporadic behavioral variant frontotemporal dementia. Linear regression analyses indicated that lower baseline respiratory sinus arrhythmia was associated with worse behavioral symptom severity and lower empathic concern and perspective-taking across the C9orf72 expansion carrier clinical spectrum. Whole-brain voxel-based morphometry analyses in participants with C9orf72 pathogenic expansions found that lower baseline respiratory sinus arrhythmia correlated with smaller gray matter volume in the left frontoinsula and bilateral thalamus, key structures that support parasympathetic function, and in the bilateral parietal lobes, occipital lobes, and cerebellum, regions that are also vulnerable in individuals with C9orf72 expansions. This study provides novel evidence that basal parasympathetic functioning is diminished in FTD due to C9orf72 expansions and suggests that baseline respiratory sinus arrhythmia may be a potential non-invasive biomarker that is sensitive to behavioral symptoms in the early stages of disease.

Project description:Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are part of a disease spectrum associated with TDP-43 pathology. Strong evidence supporting this is the existence of kindreds with family members affected by FTD, ALS or mixed features of FTD and ALS, referred to as FTD-MND. Some of these families have linkage to chromosome 9, with hexanucleotide expansion mutation in a noncoding region of C9ORF72. Discovery of the mutation defines c9FTD/ALS. Prior to discovery of mutations in C9ORF72, it was assumed that TDP-43 pathology in c9FTD/ALS was uniform. In this study, we examined the neuropathology and clinical features of 20 cases of c9FTD/ALS from a brain bank for neurodegenerative disorders. Included are six patients clinically diagnosed with ALS, eight FTD, one FTD-MND and four Alzheimer-type dementia. Clinical information was unavailable for one patient. Pathologically, the cases all had TDP-43 pathology, but there were three major pathologic groups: ALS, FTLD-MND and FTLD-TDP. The ALS cases were morphologically similar to typical sporadic ALS with almost no extramotor TDP-43 pathology; all had oligodendroglial cytoplasmic inclusions. The FTLD-MND showed predominantly Mackenzie Type 3 TDP-43 pathology, and all had ALS-like pathology in motor neurons, but more extensive extramotor pathology, with oligodendroglial cytoplasmic inclusions and infrequent hippocampal sclerosis. The FTLD-TDP cases had several features similar to FTLD-TDP due to mutations in the gene for progranulin, including Mackenzie Type 1 TDP-43 pathology with neuronal intranuclear inclusions and hippocampal sclerosis. FTLD-TDP patients were older and some were thought to have Alzheimer-type dementia. In addition to the FTD and ALS clinical presentations, the present study shows that c9FTD/ALS can have other presentations, possibly related to age of onset and the presence of hippocampal sclerosis. Moreover, there is pathologic heterogeneity not only between ALS and FTLD, but also within the FTLD group. Further studies are needed to address the molecular mechanism of clinical and pathological heterogeneity of c9FTD/ALS due to mutations in C9ORF72.

Dataset Information

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

Publications

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

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