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Updated: Jul 26, 2025
Measuring RAN Peptide Toxicity in C. elegans
Published on: April 30, 2020
Toxicity of C9orf72-associated dipeptide repeat peptides is modified by commonly used protein tags
Javier Morón-Oset1, Lilly Ks Fischer1, Mireia Carcolé2,3
1Max Planck Institute for Biology of Ageing, Cologne, Germany.
Abstract:
Hexanucleotide repeat expansions in the C9orf72 gene are the most prevalent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts of the expansions are translated into toxic dipeptide repeat (DPR) proteins. Most preclinical studies in cell and animal models have used protein-tagged polyDPR constructs to investigate DPR toxicity but the effects of tags on DPR toxicity have not been systematically explored. Here, we used Drosophila to assess the influence of protein tags on DPR toxicity. Tagging of 36 but not 100 arginine-rich DPRs with mCherry increased toxicity, whereas adding mCherry or GFP to GA100 completely abolished toxicity. FLAG tagging also reduced GA100 toxicity but less than the longer fluorescent tags. Expression of untagged but not GFP- or mCherry-tagged GA100 caused DNA damage and increased p62 levels. Fluorescent tags also affected GA100 stability and degradation. In summary, protein tags affect DPR toxicity in a tag- and DPR-dependent manner, and GA toxicity might be underestimated in studies using tagged GA proteins. Thus, including untagged DPRs as controls is important when assessing DPR toxicity in preclinical models.
Insights
Protein tags can alter the toxicity of dipeptide repeat proteins (DPRs) linked to C9orf72-associated neurodegenerative diseases. Researchers found that tags can mask or enhance DPR toxicity, impacting preclinical research findings.
Area of Science:
- Neurogenetics
- Molecular Toxicology
- Drosophila melanogaster models
Background:
- Hexanucleotide repeat expansions in the C9orf72 gene are a leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
- These expansions produce toxic dipeptide repeat (DPR) proteins, implicated in disease pathogenesis.
- Preclinical research often utilizes tagged DPR constructs, but the impact of these tags on toxicity remains poorly understood.
Purpose of the Study:
- To systematically investigate the influence of different protein tags (mCherry, GFP, FLAG) on the toxicity of C9orf72-associated DPRs in a Drosophila model.
- To determine if protein tags affect DPR stability, degradation, and associated cellular damage.
- To assess the implications of tag-induced alterations in toxicity for preclinical studies of ALS and FTD.
Main Methods:
- Expression of various tagged and untagged arginine-rich DPRs (including GA100) in Drosophila melanogaster.
- Assessment of DPR toxicity through phenotypic analysis in flies.
- Evaluation of DNA damage and p62 levels as indicators of cellular stress.
- Analysis of DPR protein stability and degradation pathways.
Main Results:
- mCherry tagging of 36-repeat DPRs increased toxicity, while mCherry or GFP tagging of GA100 abolished toxicity.
- FLAG tagging reduced GA100 toxicity, but less significantly than fluorescent tags.
- Untagged GA100, but not tagged versions, induced DNA damage and elevated p62 levels, indicating altered cellular effects.
- Fluorescent tags influenced GA100 protein stability and degradation.
Conclusions:
- Protein tags significantly impact DPR toxicity in a tag- and DPR-dependent manner.
- The toxicity of DPRs, particularly GA, may be underestimated in studies employing tagged constructs.
- Including untagged DPR controls is crucial for accurate assessment of toxicity in preclinical models of C9orf72-related neurodegenerative diseases.
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