A pH imbalance is linked to autophagic dysregulation of inner ear hair cells in Atp6v1ba-deficient zebrafish.

V-ATPase is an ATP hydrolysis-driven proton pump involved in the acidification of intracellular organelles and systemic acid-base homeostasis through H+ secretion in the renal collecting ducts. V-ATPase dysfunction is associated with hereditary distal renal tubular acidosis (dRTA). ATP6V1B1 encodes the B1 subunit of V-ATPase that is integral to ATP hydrolysis and subsequent H+ transport. Patients with pathogenic ATP6V1B1 mutations often exhibit an early onset of sensorineural hearing loss. However, the mechanisms underlying this association remain unclear. We employed morpholino oligonucleotide-mediated knockdown and CRISPR/Cas9 gene editing to generate Atp6v1ba-deficient (atp6v1ba-/-) zebrafish as an ortholog model for ATP6V1B1. The atp6v1ba-/- zebrafish exhibited systemic acidosis and significantly smaller otoliths compared to wild-type siblings. Moreover, deficiency in Atp6v1ba led to degeneration of inner ear hair cells, with ultrastructural changes indicative of autophagy. Our findings indicate a critical role of ATP6V1B1 in regulating lysosomal pH and autophagy in hair cells, and the results provide insights into the pathophysiology of sensorineural hearing loss in dRTA. Furthermore, this study demonstrates that the atp6v1ba-/- zebrafish model is a valuable tool for further investigation into disease mechanisms and potential therapies for acidosis-related hearing impairment.

Cardiac manifestations of human ACTA2 variants recapitulated in a zebrafish model.

The ACTA2 gene encodes actin α2, a major smooth muscle protein in vascular smooth muscle cells. Missense variants in the ACTA2 gene can cause inherited thoracic aortic diseases with characteristic symptoms, such as dysfunction of smooth muscle cells in the lungs, brain vessels, intestines, pupils, bladder, or heart. We identified a heterozygous missense variant of Gly148Arg (G148R) in a patient with a thoracic aortic aneurysm, dissection, and left ventricular non-compaction. We used zebrafish as an in vivo model to investigate whether or not the variants might cause functional or histopathological abnormalities in the heart. Following the fertilization of one-cell stage embryos, we injected in vitro synthesized ACTA2 mRNA of wild-type, novel variant G148R, or the previously known pathogenic variant Arg179His (R179H). The embryos were maintained and raised for 72 h post-fertilization for a heart analysis. Shortening fractions of heart were significantly reduced in both pathogenic variants. A histopathological evaluation showed that the myocardial wall of ACTA2 pathogenic variants was thinner than that of the wild type, and the total cell number within the myocardium was markedly decreased in all zebrafish with pathogenic variants mRNAs. Proliferating cell numbers were also significantly decreased in the endothelial and myocardial regions of zebrafish with ACTA2 variants compared to the wild type. These results demonstrate the effects of ACTA2 G148R and R179H on the development of left ventricle non-compaction and cardiac morphological abnormalities. Our study highlights the previously unknown significance of the ACTA2 gene in several aspects of cardiovascular development.

Behavioral and neurological effects of Vrk1 deficiency in zebrafish.

Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase, for which mutations have been reported cause to neurodegenerative diseases, including spinal muscular atrophy, characterized by microcephaly, motor dysfunction, and impaired cognitive function, in humans. Partial Vrk1 knockdown in mice has been associated with microcephaly and impaired motor function. However, the pathophysiological relationship between VRK1 and neurodegenerative disorders and the precise mechanism of VRK1-related microcephaly and motor function deficits have not been fully investigated. To address this, in this study, we established vrk1-deficient (vrk1-/-) zebrafish and found that they show mild microcephaly and impaired motor function with a low brain dopamine content. Furthermore, vrk1-/- zebrafish exhibited decreased cell proliferation, defects in nuclear envelope formation, and heterochromatin formation in the brain. To our knowledge, this is the first report demonstrating the important role of VRK1 in microcephaly and motor dysfunction in vivo using vrk1-/- zebrafish. These findings contribute to elucidating the pathophysiological mechanisms underlying VRK1-mediated neurodegenerative diseases associated with microcephaly.

Exosc2 deficiency leads to developmental disorders by causing a nucleotide pool imbalance in zebrafish.

Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2-/-) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2-/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.

Biallelic variants in LARS1 induce steatosis in developing zebrafish liver via enhanced autophagy.

BACKGROUND: Biallelic pathogenic variants of LARS1 cause infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute hepatic failure with steatosis in infants. LARS functions as a protein associated with mTORC1 and plays a crucial role in amino acid-triggered mTORC1 activation and regulation of autophagy. A previous study demonstrated that larsb-knockout zebrafish exhibit conditions resembling ILFS. However, a comprehensive analysis of larsb-knockout zebrafish has not yet been performed because of early mortality. METHODS: We generated a long-term viable zebrafish model carrying a LARS1 variant identified in an ILFS1 patient (larsb-I451F zebrafish) and analyzed the pathogenesis of the affected liver of ILFS1. RESULTS: Hepatic dysfunction is most prominent in ILFS1 patients during infancy; correspondingly, the larsb-I451F zebrafish manifested hepatic anomalies during developmental stages. The larsb-I451F zebrafish demonstrates augmented lipid accumulation within the liver during autophagy activation. Inhibition of DGAT1, which converts fatty acids to triacylglycerols, improved lipid droplets in the liver of larsb-I451F zebrafish. Notably, treatment with an autophagy inhibitor ameliorated hepatic lipid accumulation in this model. CONCLUSIONS: Our findings suggested that enhanced autophagy caused by biallelic LARS1 variants contributes to ILFS1-associated hepatic dysfunction. Furthermore, the larsb-I451F zebrafish model, which has a prolonged survival rate compared with the larsb-knockout model, highlights its potential utility as a tool for investigating the pathophysiology of ILFS1-associated liver dysfunction.

Effect of nonsense-mediated mRNA decay factor SMG9 deficiency on premature aging in zebrafish.

SMG9 is an essential component of the nonsense-mediated mRNA decay (NMD) machinery, a quality control mechanism that selectively degrades aberrant transcripts. Mutations in SMG9 are associated with heart and brain malformation syndrome (HBMS). However, the molecular mechanism underlying HBMS remains unclear. We generated smg9 mutant zebrafish (smg9oi7/oi7) that have a lifespan of approximately 6 months or longer, allowing for analysis of the in vivo function of Smg9 in adults in more detail. smg9oi7/oi7 zebrafish display congenital brain abnormalities and reduced cardiac contraction. Additionally, smg9oi7/oi7 zebrafish exhibit a premature aging phenotype. Analysis of NMD target mRNAs shows a trend toward increased mRNA levels in smg9oi7/oi7 zebrafish. Spermidine oxidase (Smox) is increased in smg9oi7/oi7 zebrafish, resulting in the accumulation of byproducts, reactive oxygen species, and acrolein. The accumulation of smox mRNA due to NMD dysregulation caused by Smg9 deficiency leads to increased oxidative stress, resulting in premature aging.