Toxicity of C9orf72-associated dipeptide repeat peptides is modified by commonly used protein tags

Hexanucleotide repeat expansions in the C9orf72 gene are most prevalent genetic cause of amyotrophic lateral sclerosis frontotemporal dementia. Transcripts of the expansions translated into toxic dipeptide repeat (DPR) proteins. preclinical studies in cell and animal models have used protein tagged polyDPR constructs to investigate DPR toxicity but effects of tags on DPR toxicity have not been systematically explored. Here, we used Drosophila to assess the influence 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. 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 Fluorescent tags also affected GA100 stability and degradation. summary, protein tags affect DPR toxicity in a tag-and dependent manner, and GA toxicity might be underestimated studies using tagged GA proteins. Thus, including untagged DPRs controls is important when assessing DPR toxicity in preclinical models.