bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.01.17.576077

RAN translation of C9ORF72-related dipeptide repeat proteins recapitulates hallmarks of motor neurone disease and identifies hypothermia as a therapeutic strategy in zebrafish

Burrows, D. J.·McGown, A.·Abduljabbar, O.·Castelli, L. M.·Hautbergue, G. M.·Ramesh, T. M.
View original source ↗Export metadata ↓

Abstract

ObjectiveHexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A large body of evidence implicates DPRs as one of the main drivers of neuronal injury in cell and animal models. MethodsA pure repeat-associated non-AUG (RAN) translation zebrafish model of C9orf72-ALS/FTD was generated. Embryonic and adult transgenic zebrafish lysates were investigated for the presence of RAN-translated DPR species and adult-onset motor deficits. Using C9orf72 cell models as well as embryonic C9orf72-ALS/FTD zebrafish, hypothermic-therapeutic temperature management (TTM) was explored as a potential therapeutic option for C9orf72-ALS/FTD. ResultsHere we describe a pure RAN translation zebrafish model of C9orf72-ALS/FTD that exhibits significant RAN-translated DPR pathology and progressive motor decline. We further demonstrate that hypothermic-TTM results in a profound reduction in DPR species in C9orf72-ALS/FTD cell models as well as embryonic C9orf72-ALS/FTD zebrafish. InterpretationThe transgenic model detailed in this paper provides a medium throughput in vivo research tool to further investigate the role of RAN-translation in C9orf72-ALS/FTD and further understand the mechanisms that underpin neuroprotective strategies. Hypothermic-TTM presents a viable therapeutic avenue to explore in the context of C9orf72-ALS/FTD.

Source connections

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Burrows, D. J., McGown, A., Abduljabbar, O., Castelli, L. M., Hautbergue, G. M., Ramesh, T. M.. 2024-01-20. RAN translation of C9ORF72-related dipeptide repeat proteins recapitulates hallmarks of motor neurone disease and identifies hypothermia as a therapeutic strategy in zebrafish. https://doi.org/10.1101/2024.01.17.576077

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Plasmid architecture determines the stability of inverted terminal repeats in adeno-associated virus vectors

Recombinant vectors derived from adeno-associated viruses (rAAVs) are a mainstay of human gene therapy. rAAVs are produced from plasmids containing transgene cassettes flanked by inverted terminal repeats (ITRs), which form structured DNA elements that stabilize the ends of the single-stranded viral genome and are the only viral sequences required in cis for genome packaging. For decades, it has been recognized that propagation of ITR-containing plasmids can result in deletions and other mutations, prompting the use of specialized bacterial strains, modified growth conditions, and truncated or altered ITRs. Despite these practices, ITR instability remains a persistent source of plasmid heterogeneity. To identify determinants of ITR stability, we evaluated ITR integrity in one of the original cloned AAV2 genome isolates, a reconstructed AAV2 plasmid, and a synthetic rAAV vector containing full-length native AAV2 ITRs. We established a quantitative bioinformatic workflow for analyzing ITR-containing plasmids and virus preparations from raw Oxford Nanopore sequencing data. These experiments showed that ITRs were highly stable during short-term culture, whereas prolonged culture revealed strong positional effects, with preferential loss or mutation of the ITR nearest the plasmid origin of replication. Consistent with this model, a survey of 7,041 sequence-verifiable AAV plasmids from the Addgene repository identified a widely disseminated 11-bp ITR deletion in 4,773 plasmids; among analyzable two-ITR plasmids, this deletion was located in the origin-proximal ITR in 95.3% of cases. Guided by these findings, we constructed a novel rAAV entry vector with stable full-length native AAV2 ITRs that enabled efficient packaging of a 4,750-bp all-in-one CRISPR-Cas9 cassette. Finally, we developed a cell-based strategy to compare the effects of ITR mutations on rAAV genome integration, providing preliminary evidence that ITR sequence variation can influence integration outcomes. Together, these findings show that ITR instability is a preventable, position-dependent property of plasmid architecture and identify ITR integrity as an important variable in rAAV vector design and quality control.

molecular biology↗
bioRxiv2026

Single-point mutation alters odorant receptor sensitivity associated with host plant specialization in Spodoptera moths

Host specialization in herbivorous insects is often associated with divergence in chemosensory abilities. Here, we investigated the possible contribution of odorant receptors (ORs) in host plant restriction in the lily moth Spodoptera picta, a species specialized on Amaryllidaceae. Manual annotation of S. picta ORs in its genome revealed a repertoire similar in size and composition to those of its polyphagous sister species, S. littoralis and S. litura, suggesting that specialization did not involve major gene loss or expansion in the lily moth. To assess functional divergence beyond gene number, we applied a large scaled structure-based virtual screening approach to the entire OR repertoires of these three Spodoptera species, generating ligand-binding profiles for 120,591 volatile compounds. Among 69 1:1:1 OR orthologs, 24 exhibited divergent predicted binding spectra. We pinpointed OR29 that we also found to be highly expressed in both male and female antennae of S. picta through a RNAseq approach. Functional assays demonstrated that S. picta OR29 acquired heightened sensitivity to limonene enantiomers, volatiles emitted by host Amaryllidaceae inflorescences. Site-directed mutagenesis revealed that a single amino acid substitution within the predicted binding region underlies this shift in sensitivity. These results show that host specialization in S. picta has not been accompanied by significant OR repertoire remodeling, but rather by subtle molecular changes that fine-tune receptor sensitivity to host-derived volatiles.

molecular biology↗
bioRxiv2026

Arc represses gene expression in IS605-family transposons

Bacterial insertion sequences (IS) are compact transposable elements that encode proteins required for their mobility and maintenance, yet many also encode accessory proteins with poorly understood functions. For example, IS605-family elements often encode a transposase called TnpA and an RNA-guided nuclease called TnpB that supports transposon maintenance, alongside an additional ribbon-helix-helix protein named Arc. Though the roles of TnpA and TnpB have been extensively studied in recent years, the enigmatic function of Arc has not been investigated. Here, we show that Arc acts as a transcriptional repressor to directly bind the transposon's native promoter sequence regulating TnpA and TnpB gene expression. By systematically testing Arc-containing IS605 elements, we identified a conserved binding pattern at intergenic transposon sequences neighboring protein-coding genes through chromatin immunoprecipitation and sequencing analyses. We then used fluorescence reporter assays and demonstrated that these intergenic sequences function as strong promoters, and that the presence of Arc dramatically reduces their gene expression. Together, these findings identify Arc as a transposon-encoded transcriptional repressor, revealing a regulatory layer that may promote long-term persistence of IS605-family elements by keeping their activity in check. The widespread association of Arc homologs with diverse mobile elements and cellular genes suggests that these compact regulators may more broadly restrain the expression of neighboring genetic machinery across varied genomic contexts. neighboring genetic machinery across varied genomic contexts.

molecular biology↗