Characterization of a C9orf72 KnockoutDanio reriomodel for ALS and cross-species validation of potential therapeutics screened inCaenorhabditis elegans
Abstract
Intronic hexanucleotide repeat expansions in theC9orf72gene represent the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This expansion decreasesC9orf72expression in affected patients, indicating that loss ofC9orf72function (LOF) acts as a pathogenic mechanism. Several models usingDanio rerio(zebrafish) forC9orf72depletion have been developed to explore disease mechanisms and the consequences ofC9orf72LOF. However, inconsistencies exist in reported phenotypes, and many have yet to be validated in stable germline ablation models. To address this, we created a zebrafishC9orf72knockout model using CRISPR/Cas9. TheC9orf72LOF model demonstrates, in a generally dose-dependent manner, increased larval mortality, persistent growth reduction, and motor deficits. Additionally, homozygousC9orf72LOF larvae exhibited mild overbranching of spinal motoneurons. To identify potential therapeutic compounds, we performed a screen on an establishedCaenorhabditis elegans(C. elegans)C9orf72homologue (alfa-1) LOF model, identifying 12 compounds that enhanced motility, reduced neurodegeneration, and alleviated paralysis phenotypes. Motivated by the shared motor phenotype, 2 of those compounds were tested in our zebrafishC9orf72LOF model. Pizotifen malate was found to significantly improve motor deficits inC9orf72LOF zebrafish larvae. We introduce a novel zebrafishC9orf72knockout model that exhibits phenotypic differences from depletion models, providing a valuable tool forin vivo C9orf72research and ALS therapeutic validation. Furthermore, we identify pizotifen malate as a promising compound for further preclinical evaluation.
Author Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive loss of motor neurons, with no curative treatments currently available. The most common genetic cause is a hexanucleotide repeat expansion in theC9orf72gene, which reduces its expression and implicates loss-of-function (LOF) as a disease mechanism. However, the complete functions of C9orf72 and its role in ALS remain unclear. Zebrafish models with indirect partial reduction ofC9orf72expression have shown promise in recapitulating key aspects of ALS, but inconsistencies have been observed across these models. To address these challenges, we developed a stable geneticC9orf72LOF zebrafish model to study the effects of its LOF, validate previous findings, and test potential ALS therapeutics. Our model displays swimming activity deficits, reduced growth, increased mortality, and mild spinal motor neuron abnormalities. We demonstrated that pizotifen malate significantly improved motor function in both our model and a similar well-established worm model. These results underscore the differences between indirect depletion and direct genetic LOF models while identifying pizotifen malate as a promising candidate for preclinical testing. This zebrafish model serves as a valuable tool for understandingC9orf72-associated ALS mechanisms and advancing therapeutic development.
Related articles
Related articles are currently not available for this article.