ALS gene, C9orf72, loss of function Zebrafish model shows motor and synaptic defects

Zoé Butti¹, Jean Giacomotto² & Kessen Patten¹

¹INRS-Institut Armand-Frappier, National Institute of Scientific Research, Laval, QC, Canada
² Queensland Brain Institute, The University of Queensland, Brisbane, QLD 4072, Australia; Institut NeuroMyoGene, CNRS UMR5310, INSERM U1217, Universite Claude Bernard Lyon 1, 69622 Villeurbanne, France.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motoneurons causing muscular atrophy, paralysis and ultimately death. Presently, no curative treatment exists. Understanding the physiopathological mechanisms will help develop new efficient treatments. In 2011, an expansion of a repetition of a hexanucleotide (GGGGCC) in the first intronic region of the C9orf72 gene has been discovered as the first genetic cause of ALS. To investigate the role of C9orf72 loss of function in ALS, we used synthetic micro-RNAs to specifically target the zebrafish C9orf72 gene (C9-miRNA) and have developed a stable zebrafish C9-miRNA line with reduced expression of C9orf72. Upon loss of function of C9orf72, we observed that zebrafish C9-miRNA mutants display severe motor deficits beginning 6 days postfertilization (6 dpf) and a majority die prematurely at 15 dpf. Analysis of the neuromuscular junctions using specific presynaptic and postsynaptic markers SV2 and alpha-bungarotoxin respectively, revealed a significant decrease in the number of synaptic contacts in the C9-miRNA line at 6 dpf correlating with a decreased synaptic vesicles turnover. Electrophysiology recordings using patch clamp technique on muscle fibres showed a decrease of amplitude and frequency of the spontaneous miniature end plate currents, which suggests a decrease number of presynaptic endings. Also, TDP-43 has been shown to aggregates at 6 dpf in our C9-miRNA. Among the few fishes that survived until adulthood, we observed a significant motoneuron and muscle atrophy. Altogether, our zebrafish C9-miRNA replicates aspects of ALS and showed that C9orf72 has a role in the synaptic transmission at the NMJ.