The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.

Amyotrophic lateral sclerosis (ALS) presents clinically in adulthood and is characterized by the loss of motoneurons in the spinal cord and cerebral cortex. Animal models of the disease suggest that significant neuronal abnormalities exist during preclinical stages of the disease. Mutations in the gene fused in sarcoma (FUS) are associated with ALS and cause impairment in motor function in animal models. However, the mechanism of neuromuscular dysfunction underlying pathophysiological deficits causing impairment in locomotor function resulting from mutant FUS expression is unknown. To characterize the cellular pathophysiological defect, we expressed the wild-type human gene (wtFUS) or the ALS-associated mutation R521H (mutFUS) gene in zebrafish larvae and characterized their motor (swimming) activity and function of their neuromuscular junctions (NMJs). Additionally, we tested knockdown of zebrafish fus with an antisense morpholino oligonucleotide (fus AMO). Expression of either mutFUS or knockdown of fus resulted in impaired motor activity and reduced NMJ synaptic fidelity with reduced quantal transmission. Primary motoneurons expressing mutFUS were found to be more excitable. These impairments in neuronal function could be partially restored in fus AMO larvae also expressing wtFUS (fus AMO+wtFUS) but not mutFUS (fus AMO+mutFUS). These results show that both a loss and gain of FUS function result in defective presynaptic function at the NMJ.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C . elegans , validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C . elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca ²⁺ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease.

Cortical spreading depression (CSD) is closely associated with important pathologies including stroke, seizures and migraine. The mechanisms underlying SD in its various forms are still incompletely understood. Here we describe SD-like events in an invertebrate model, the ventilatory central pattern generator (CPG) of locusts. Using K⁺ -sensitive microelectrodes, we measured extracellular K⁺ concentration ([K⁺]_o) in the metathoracic neuropile of the CPG while monitoring CPG output electromyographically from muscle 161 in the second abdominal segment to investigate the role K⁺ in failure of neural circuit operation induced by various stressors. Failure of ventilation in response to different stressors (hyperthermia, anoxia, ATP depletion, Na⁺/K⁺ ATPase impairment, K⁺ injection) was associated with a disturbance of CNS ion homeostasis that shares the characteristics of CSD and SD-like events in vertebrates. Hyperthermic failure was preconditioned by prior heat shock (3 h, 45°C) and induced-thermotolerance was associated with an increase in the rate of clearance of extracellular K⁺ that was not linked to changes in ATP levels or total Na⁺/K⁺ ATPase activity. Our findings suggest that SD-like events in locusts are adaptive to terminate neural network operation and conserve energy during stress and that they can be preconditioned by experience. We propose that they share mechanisms with CSD in mammals suggesting a common evolutionary origin.

TAR DNA binding protein (TDP-43, encoded by theTARDBPgene) has recently been shown to be associated with amyotrophic lateral sclerosis (ALS), but the early pathophysiological deficits causing impairment in motor function are unknown. Here we expressed the wild-type human gene (wtTARDBP) or the ALS mutation G348C (mutTARDBP) in zebrafish larvae and characterized their motor (swimming) activity and the structure and function of their neuromuscular junctions (NMJs). Of these groups only mutTARDBPlarvae showed impaired swimming and increased motoneuron vulnerability with reduced synaptic fidelity, reduced quantal transmission, and more orphaned presynaptic and postsynaptic structures at the NMJ. Remarkably, all behavioral and cellular features were stabilized by chronic treatment with either of the L-type calcium channel agonists FPL 64176 or Bay K 8644. These results indicate that expression of mutTARDBPresults in defective NMJs and that calcium channel agonists could be novel therapeutics for ALS.