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.