Loss of mitochondrial Chchd10 or Chchd2 in zebrafish leads to an ALS-like phenotype and Complex I deficiency independent of the mitochondrial integrated stress response.

Mutations in CHCHD10 and CHCHD2, encoding two paralogous mitochondrial proteins, have been identified in cases of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Parkinson's disease. Their role in disease is unclear, though both have been linked to mitochondrial respiration and mitochondrial stress responses. Here, we investigated the biological roles of these proteins during vertebrate development using knockout (KO) models in zebrafish. We demonstrate that loss of either or both proteins leads to motor impairment, reduced survival and compromised neuromuscular junction integrity in larval zebrafish. Compensation by Chchd10 was observed in the chchd2-/- model, but not by Chchd2 in the chchd10-/- model. The assembly of mitochondrial respiratory chain Complex I was impaired in chchd10-/- and chchd2-/- zebrafish larvae, but unexpectedly not in a double chchd10-/- and chchd2-/- model, suggesting that reduced mitochondrial Complex I cannot be solely responsible for the observed phenotypes, which are generally more severe in the double KO. We observed transcriptional activation markers of the mitochondrial integrated stress response (mt-ISR) in the double chchd10-/- and chchd2-/- KO model, suggesting that this pathway is involved in the restoration of Complex I assembly in our double KO model. The data presented here demonstrates that the Complex I assembly defect in our single KO models arises independently of the mt-ISR. Furthermore, this study provides evidence that both proteins are required for normal vertebrate development.

Development of an endogenously myc-tagged TARDBP (TDP-43) zebrafish model using the CRISPR/Cas9 system and homology directed repair.

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.

Efficacy of Ciprofloxacin/Celecoxib combination in zebrafish models of amyotrophic lateral sclerosis.

OBJECTIVE: To evaluate the efficacy of a fixed-dose combination of two approved drugs, Ciprofloxacin and Celecoxib, as a potential therapeutic treatment for amyotrophic lateral sclerosis (ALS). METHODS: Toxicity and efficacy of Ciprofloxacin and Celecoxib were tested, each alone and in distinct ratio combinations in SOD1 G93R transgenic zebrafish model for ALS. Quantification of swimming measures following stimuli, measurements of axonal projections from the spinal cord, neuromuscular junction structure and morphometric analysis of microglia cells were performed in the combination- treated vs nontreated mutant larvae. Additionally, quantifications of touch-evoked locomotor escape response were conducted in treated vs nontreated zebrafish expressing the TARDBP G348C ALS variant. RESULTS: When administered individually, Ciprofloxacin had a mild effect and Celecoxib had no therapeutic effect. However, combined Ciprofloxacin and Celecoxib (Cipro/Celecox) treatment caused a significant increase of ~ 84% in the distance the SOD1 G93R transgenic larvae swam. Additionally, Cipro/Celecox elicited recovery of impaired motor neurons morphology and abnormal neuromuscular junction structure and preserved the ramified morphology of microglia cells in the SOD1 mutants. Furthermore, larvae expressing the TDP-43 mutation displayed evoked touch responses that were significantly longer in swim distance (110% increase) and significantly higher in maximal swim velocity (~44% increase) when treated with Cipro/Celecox combination. INTERPRETATION: Cipro/Celecox combination improved locomotor and cellular deficits of ALS zebrafish models. These results identify this novel combination as effective, and may prove promising for the treatment of ALS.

Augmentation of spinal cord glutamatergic synaptic currents in zebrafish primary motoneurons expressing mutant human TARDBP (TDP-43).

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.