Implementation of an antibody characterization process: Application to the major ALS/FTD disease gene C9ORF72

Laflamme, Carl; McKeever, Paul M.; Kumar, Rahul; Schwartz, Julie; Kolahdouzan, Mahshad; Chen, Carol X.-Q.; You, Zhipeng; Benaliouad, Faïza; Gileadi, O.; McBride, Heidi M.; Durcan, Thomas M.; Edwards, A.M.; Healy, Luke M.; Robertson, Janice; McPherson, Peter S.

doi:10.1101/499350

Cited by 2 publications

(1 citation statement)

References 32 publications

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“…Numerous C9orf72 antibodies (including polyclonal, monoclonal and recombinant) have been developed, but remain largely unverified. Interestingly, a novel powerful CRISPR genome-editing approach validated multiple commercially available C9orf72 antibodies, which could be of use and may be considered for future C9orf72 lossof-function studies [62].…”

Section: C9 Patient-derived Modelsmentioning

confidence: 99%

C9orf72 loss-of-function: a trivial, stand-alone or additive mechanism in C9 ALS/FTD?

2020

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A repeat expansion in C9orf72 is responsible for the characteristic neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in a still unresolved manner. Proposed mechanisms involve gain-of-functions, comprising RNA and protein toxicity, and loss-of-function of the C9orf72 gene. Their exact contribution is still inconclusive and reports regarding loss-of-function are rather inconsistent. Here, we review the function of the C9orf72 protein and its relevance in disease. We explore the potential link between reduced C9orf72 levels and disease phenotypes in postmortem, in vitro, and in vivo models. Moreover, the significance of loss-of-function in other non-coding repeat expansion diseases is used to clarify its contribution in C9orf72 ALS/FTD. In conclusion, with evidence pointing to a multiple-hit model, loss-of-function on itself seems to be insufficient to cause neurodegeneration in C9orf72 ALS/FTD.

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Section: C9 Patient-derived Modelsmentioning

confidence: 99%

C9orf72 loss-of-function: a trivial, stand-alone or additive mechanism in C9 ALS/FTD?

2020

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Synaptic localization of C9orf72 regulates post-synaptic glutamate receptor 1 levels

Xiao

McKeever

Lau

et al. 2019

acta neuropathol commun

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A hexanucleotide repeat expansion in a noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reduction of select or total C9orf72 transcript and protein levels is observed in postmortem C9-ALS/FTD tissue, and loss of C9orf72 orthologues in zebrafish and C. elegans results in motor deficits. However, how the reduction in C9orf72 in ALS and FTD might contribute to the disease process remains poorly understood. It has been shown that C9orf72 interacts and forms a complex with SMCR8 and WDR41, acting as a guanine exchange factor for Rab GTPases. Given the known synaptosomal compartmentalization of C9orf72-interacting Rab GTPases, we hypothesized that C9orf72 localization to synaptosomes would be required for the regulation of Rab GTPases and receptor trafficking. This study combined synaptosomal and post-synaptic density preparations together with a knockout-confirmed monoclonal antibody for C9orf72 to assess the localization and role of C9orf72 in the synaptosomes of mouse forebrains. Here, we found C9orf72 to be localized to both the pre- and post-synaptic compartment, as confirmed by both post-synaptic immunoprecipitation and immunofluorescence labelling. In C9orf72 knockout (C9-KO) mice, we demonstrated that pre-synaptic Rab3a, Rab5, and Rab11 protein levels remained stable compared with wild-type littermates (C9-WT). Strikingly, post-synaptic preparations from C9-KO mouse forebrains demonstrated a complete loss of Smcr8 protein levels, together with a significant downregulation of Rab39b and a concomitant upregulation of GluR1 compared with C9-WT mice. We confirmed the localization of Rab39b downregulation and GluR1 upregulation to the dorsal hippocampus of C9-KO mice by immunofluorescence. These results indicate that C9orf72 is essential for the regulation of post-synaptic receptor levels, and implicates loss of C9orf72 in contributing to synaptic dysfunction and related excitotoxicity in ALS and FTD.

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Implementation of an antibody characterization process: Application to the major ALS/FTD disease gene C9ORF72

Cited by 2 publications

References 32 publications

C9orf72 loss-of-function: a trivial, stand-alone or additive mechanism in C9 ALS/FTD?

C9orf72 loss-of-function: a trivial, stand-alone or additive mechanism in C9 ALS/FTD?

Synaptic localization of C9orf72 regulates post-synaptic glutamate receptor 1 levels

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