Minimal Dataset for Characterization of a C9orf72 Knockout Danio rerio Model for ALS and Cross-Species Validation of Therapeutics in Caenorhabditis elegans

Intronic hexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This expansion reduces C9orf72 expression in affected patients, implicating loss of C9orf72 function (LOF) as a pathogenic mechanism. Various Danio rerio (zebrafish) models of C9orf72 depletion have been developed to investigate disease mechanisms and the effects of C9orf72 LOF.

However, there are inconsistencies in reported phenotypes, and most have yet to be validated in stable germline ablation models. To address this, we generated a zebrafish C9orf72 knockout model using CRISPR/Cas9. The C9orf72 LOF model exhibits, in a generally dose-dependent manner, increased larval mortality, persistent growth reduction, and motor deficits.

Additionally, homozygous C9orf72 LOF larvae displayed mild overbranching of spinal motoneurons. To identify potential therapeutic compounds, we conducted a screen in an established Caenorhabditis elegans (C. elegans) C9orf72 homologue (alfa-1) LOF model, identifying 12 compounds that improved the motility, neurodegeneration, and paralysis phenotypes. Prompted by the shared motor phenotype, 2 of those compounds were tested in our zebrafish C9orf72 LOF model.

Pizotifen malate was found to significantly improve motor deficits in C9orf72 LOF zebrafish larvae. We present a novel zebrafish C9orf72 knockout model that exhibits phenotypic differences from depletion models, providing a valuable tool for in vivo C9orf72 research and ALS therapeutic validation. Furthermore, we identify pizotifen malate as a promising compound for further preclinical evaluation.

Dataset Information: This dataset includes raw and processed numerical data from key experimental assays in zebrafish (Danio rerio) and Caenorhabditis elegans. In zebrafish, it contains data from qPCR, sequencing, western blots, survival assays, motor activity tracking, spinal motor neuron morphology analysis, neuromuscular junction integrity evaluation, and drug screening experiments. For C. elegans, it includes data on swimming activity, paralysis assays, neurodegeneration analysis, and drug screening experiments.

This dataset represents the minimal dataset required for reproducibility in accordance with PLOS Genetics guidelines and provides all necessary numerical data to replicate the study’s findings. Where relevant, sample raw files are included to ensure data provenance and reproducibility.