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Many of the modern advances in cellular biology have been made by the expression of engineered constructs with epitope tags for subsequent biochemical investigations. While the utility of epitope tags has permitted insights in cellular and animal models, these are often expressed using traditional transgenic approaches. Using the CRISPR/Cas9 system and homology directed repair we recombine a single myc epitope sequence following the start codon of the zebrafish ortholog of TARDBP (TDP-43). TDP-43 is an RNA binding protein that is involved in the neurodegenerative disease amyotrophic lateral sclerosis and frontotemporal dementia. We report that zebrafish expressing the myc-tardbp engendered allele produced a stable protein that was detected by both western blot and immunofluorescence. Furthermore, both heterozygous and homozygous carriers of the myc-tardbp allele developed to sexual maturity. We propose that the methodology used here will be useful for zebrafish researchers and other comparative animal biologists interested in developing animal models expressing endogenously tagged proteins., (Copyright © 2022. Published by Elsevier Inc.)

Though there is compelling evidence that de-innervation of neuromuscular junctions (NMJ) occurs early in amyotrophic lateral sclerosis (ALS), defects arising at synapses in the spinal cord remain incompletely understood. To investigate spinal cord synaptic dysfunction, we took advantage of a zebrafish larval model and expressed either wild type human TARDBP (wtTARDBP) or the ALS-causing G348C variant (mutTARDBP). The larval zebrafish is ideally suited to examine synaptic connectivity between descending populations of neurons and spinal cord motoneurons as a fully intact spinal cord is preserved during experimentation. Here we provide evidence that the tail-beat motor pattern is reduced in both frequency and duration in larvae expressing mutTARDBP. In addition, we report that motor-related synaptic depolarizations in primary motoneurons of the spinal cord are shorter in duration and fewer action potentials are evoked in larvae expressing mutTARDBP. To more thoroughly examine spinal cord synaptic dysfunction in our ALS model, we isolated AMPA/kainate-mediated glutamatergic miniature excitatory post-synaptic currents in primary motoneurons and found that in addition to displaying a larger amplitude, the frequency of quantal events was higher in larvae expressing mutTARDBP when compared to larvae expressing wtTARDBP. In a final series of experiments, we optogenetically drove neuronal activity in the hindbrain and spinal cord population of descending ipsilateral glutamatergic interneurons (expressing Chx10) using the Gal4-UAS system and found that larvae expressing mutTARDBP displayed abnormal tail-beat patterns in response to optogenetic stimuli and augmented synaptic connectivity with motoneurons. These findings indicate that expression of mutTARDBP results in functionally altered glutamatergic synapses in the spinal cord.

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development., (© 2014. Published by The Company of Biologists Ltd.)

TAR DNA binding protein (TDP-43) is a 43 kD, predominately nuclear, protein involved in RNA metabolism. Of clinical significance is that the majority of amyotrophic lateral sclerosis (ALS) patients display abnormal accumulation of misfolded TDP-43 in the cytoplasm, which is coincident with a loss of nuclear localization in the afflicted regions of the central nervous system. Little is known about defects that arise in loss-of-function models, in particular synaptic defects that arise at the neuromuscular junction (NMJ). In this report, we examined abnormalities arising at the NMJ following depletion of tdp-43 using a previously characterized mutant tardbp (encoding tdp-43) zebrafish line containing a premature stop codon (Y220X) that results in an unstable and degraded protein. Homozygous tardbpY220X/Y220X zebrafish do not produce tdp-43 but develop normally due to expression of an alternative splice variant of tardbpl (tardbp paralog). Using an antisense morpholino oligonucleotide to knockdown expression of the tardbpl in tardbpY220X/Y220X embryos, we examined locomotor defects, NMJ structural abnormalities and release of quantal synaptic vesicles at the NMJ. As in previous reports, larvae depleted of tdp-43 display reduced survival, gross morphological defects and severely impaired locomotor activity. These larvae also displayed an increased number of orphaned pre- and postsynaptic NMJ markers but surprisingly, we observed a significant increase (3.5 times) in the frequency of quantal acetylcholine release at the NMJ in larvae depleted of tdp-43. These results indicate that reduced TDP-43 levels alter quantal vesicle release at the NMJ during vertebrate development and may be relevant for understanding synaptic dysfunction in ALS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]