Transcriptome analysis of atad3-null zebrafish embryos elucidates possible disease mechanisms

ATAD3A is a nuclear gene encoding the ATAD3A protein which has diverse roles in the mitochondria, affecting various cellular processes ranging from mitochondrial dynamics and mtDNA maintenance to metabolism pathways and mitochondrial interactions with other organelles. Pathogenic variants in this gene cause neurological diseases in humans with recognizable genotype-phenotype correlations. Using CRISPR/Cas9 genome editing, we generated a knockout (KO) model for the ortholog gene atad3 in zebrafish., in order to further characterize the gene and its role in health and disease. We examined the phenotype, mitochondrial content and mitochondrial activity of the mutant embryos, and compared the transcriptome of wild-type and mutant embryos at 3 days post-fertilization (dpf) via RNAseq. Results were validated by quantitative real-time PCR at 3dpf and 5dpf. Atad3-null zebrafish embryos demonstrated microcephaly, small eyes, pericardial edema and thinning of the musculature, closely correlating with the human disease. Mitochondrial content and activity were reduced. Transcriptome analysis revealed an expected decline in most mitochondrial pathways in the mutant embryos. In addition, we witnessed a global upregulation of cytosolic tRNA synthetases, presumably secondary to mitochondrial stress and possibly endoplasmic reticulum (ER)-stress. Differential expression of select genes was confirmed in fibroblasts from an affected individual. Zebrafish atad3-null embryos can be used as a reliable model of human ATAD3A-associated disorders, as the pathways and processes differentially expressed in this model are similar to the ones in humans. Moreover, our study confirms mitochondrial dysfunction as the primary underlying pathogenic mechanism in ATATD3A-associated disorders. Overall design: Using CRISPR/cas9 gene editing technology, we created a zebrafish line heterozygous for atad3 deletion. Embryos of two heterozygous fish were collected at 3 days post fertilization (dpf), when phenotype begins to be apparent, for DNA and RNA extraction. DNA was used for genotyping and RNA was pooled according to genotype. Transcriptome compares siblings, wild-type and atad3-null embryos.