Dataset Information

Mutations to the ALS/FTD gene CCNF reveals elevated Lys48-linked ubiquitination by the SCF(cyclin F) complex

ABSTRACT: Approximately 10% of Amyotrophic lateral sclerosis (ALS) cases have a positive family history (familial ALS) and appear clinically indistinguishable from sporadic cases. ALS and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics; however, their biological mechanisms remain poorly understood. We have previously identified CCNF missense mutations in cohorts of familial and sporadic ALS and FTD cases [ref]. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFCyclin F) complex that is responsible for ubiquitinating proteins for degradation by the Ubiquitin-Proteasome System (UPS). The SCFCyclin F complex is essential for maintaining cellular homeostasis in cells; but mutations appear to lead to aberrant motor neuron development and neuron degeneration. We revealed elevated Lys48-specific ubiquitination of proteins in neuronal cells expressing mutant CCNFS621G compared to the CCNFWT control, which is consistent with increased Lys48-specific E3 ligase activity of cyclin FS621G (>1.3-fold). Different subsets of immunoprecipitated Lys48-ubiquitinated proteins were identified between CCNFWT and CCNFS621G cells indicating that transfected cyclin F contribute to the protein ubiquitination profile. These findings provide mechanistic insights into the effects of CCNF gene mutations on the function of the SCFcyclin F complex on neuronal proteostasis.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Neuroblast

DISEASE(S): Amyotrophic Lateral Sclerosis

SUBMITTER: Albert Lee

LAB HEAD: Albert Lee

PROVIDER: PXD002011 | Pride | 2017-12-01

REPOSITORIES: Pride

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Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy.

Lee Albert A Rayner Stephanie L SL Gwee Serene S L SSL De Luca Alana A Shahheydari Hamideh H Sundaramoorthy Vinod V Ragagnin Audrey A Morsch Marco M Radford Rowan R Galper Jasmin J Freckleton Sarah S Shi Bingyang B Walker Adam K AK Don Emily K EK Cole Nicholas J NJ Yang Shu S Williams Kelly L KL Yerbury Justin J JJ Blair Ian P IP Atkin Julie D JD Molloy Mark P MP Chung Roger S RS

Cellular and molecular life sciences : CMLS 20170829 2

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCF<sup>cyclin F</sup>) complex that is ...[more]

PMID: 28852778

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Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. RNA was extracted from fibroblasts grown from neurologically healthy controls (n=6) and 3 groups of patients with ALS: 1) sporadic cases (n=6); 2) cases due to mutations of SOD1 (n=4); 3) cases due to mutations of TARDBP (n=3). The three ALS groups were compared to the controls.

Dataset Information

Mutations to the ALS/FTD gene CCNF reveals elevated Lys48-linked ubiquitination by the SCF(cyclin F) complex

Publications

Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy.

Similar Datasets

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