Human CLP1 mutations alter tRNA biogenesis, affecting both peripheral and central nervous system function.

CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.

DHCR7 links cholesterol synthesis with neuronal development and axonal integrity.

The DHCR7 enzyme converts 7-DHC into cholesterol. Mutations in DHCR7 can block cholesterol production, leading to abnormal accumulation of 7-DHC and causing Smith-Lemli-Opitz syndrome (SLOS). SLOS is an autosomal recessive disorder characterized by multiple malformations, including microcephaly, intellectual disability, behavior reminiscent of autism, sleep disturbances, and attention-deficit/hyperactivity disorder (ADHD)-like hyperactivity. Although 7-DHC affects neuronal differentiation in ex vivo experiments, the precise mechanism of SLOS remains unclear. We generated Dhcr7 deficient (dhcr7-/-) zebrafish that exhibited key features of SLOS, including microcephaly, decreased neural stem cell pools, and behavioral phenotypes similar to those of ADHD-like hyperactivity. These zebrafish demonstrated compromised myelination, synaptic anomalies, and neurotransmitter imbalances. The axons of the dhcr7-/- zebrafish showed increased lysosomes and attenuated autophagy, suggesting that autophagy-related neuronal homeostasis is disrupted.

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.

Vrk2 deficiency elicits aggressive behavior in female zebrafish.

Vaccinia-related kinase 2 (VRK2) is a serine/threonine kinase initially identified in highly proliferative cells such as thymocytes and fetal liver cells, and it is involved in cell proliferation and survival. VRK2 is also expressed in the brain; however, its molecular function in the central nervous system is mostly unknown. Many genome-wide association studies (GWASs) have reported that VRK2 is a potential candidate molecule for neuropsychiatric diseases such as schizophrenia in humans. However, the pathophysiological relationship between VRK2 and neuropsychiatric disorders has not been fully investigated. In this study, we evaluated vrk2-deficient (vrk2-/- ) zebrafish and found that vrk2-/- female zebrafish showed aggressive behavior and different social preference compared with control (vrk2+/+ ) zebrafish, with low gamma-aminobutyric acid (GABA) content in the brain and high density of neuronal dendrites when compared to vrk2+/+ zebrafish. These findings suggest that female vrk2-/- zebrafish were indeed a model of malbehavior characterized by aggression and social interaction, which can be attributed to the low levels of GABA content in their brain.

Ankle2 deficiency-associated microcephaly and spermatogenesis defects in zebrafish are alleviated by heterozygous deletion of vrk1.

Autosomal recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by a below average brain volume at birth and is associated with neurodevelopmental disorders such as growth retardation and intellectual disability. Mutations in ANKLE2 have been identified as one of the causes of MCPH (MCPH16). ANKLE2 is a target molecule of the Zika virus NS4a protein that interferes with ANKLE2 function, resulting in severe microcephaly. ANKLE2 is essential for organizing the nuclear envelope and chromatin structures during the mitotic-end process via barrier to autointegration factor (BAF) dephosphorylation. However, the precise mechanism by which the loss of ANKLE2 function causes the pathogenesis of microcephaly remains unclear. In this study, we generated Ankle2-deficient zebrafish (ankle2-/-) with a significant reduction in brain size compared with that of their control siblings. The ankle2-/- brain showed a significant decrease in the number of radial glial progenitor cells, suggesting that Ankle2 deficiency in zebrafish causes neurogenesis defects. Furthermore, ankle2-/- male zebrafish showed infertility owing to defects in spermatogenesis. Notably, microcephaly was overcome by vrk1 morpholino knockdown or vrk1 heterozygous deletion. In addition, spermatogenesis in ankle2-/- zebrafish males was partially restored by the vrk1 heterozygous deletion, although infertility was not resolved. These results indicate that ANKLE2 and VRK1 coordinate with each other for BAF phosphorylation to maintain normal mitosis during neurogenesis and spermatogenesis.

Modeling a human CLP1 mutation in mouse identifies an accumulation of tyrosine pre-tRNA fragments causing pontocerebellar hypoplasia type 10.

Cleavage factor polyribonucleotide kinase subunit 1 (CLP1), an RNA kinase, plays essential roles in protein complexes involved in the 3'-end formation and polyadenylation of mRNA and the tRNA splicing endonuclease complex, which is involved in precursor tRNA splicing. The mutation R140H in human CLP1 causes pontocerebellar hypoplasia type 10 (PCH10), which is characterized by microcephaly and axonal peripheral neuropathy. Previously, we reported that RNA fragments derived from isoleucine pre-tRNA introns (Ile-introns) accumulate in fibroblasts of patients with PCH10. Therefore, it has been suggested that this intronic RNA fragment accumulation may trigger PCH10 onset. However, the molecular mechanism underlying PCH10 pathogenesis remains elusive. Thus, we generated knock-in mutant mice that harbored a CLP1 mutation consistent with R140H. As expected, these mice showed progressive loss of the upper motor neurons, resulting in impaired locomotor activity, although the phenotype was milder than that of the human variant. Mechanistically, we found that the R140H mutation causes intracellular accumulation of Ile-introns derived from isoleucine pre-tRNAs and 5' tRNA fragments derived from tyrosine pre-tRNAs, suggesting that these two types of RNA fragments were cooperatively or independently involved in the onset and progression of the disease. Taken together, the CLP1-R140H mouse model provided new insights into the pathogenesis of neurodegenerative diseases, such as PCH10, caused by genetic mutations in tRNA metabolism-related molecules.

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.

Tyrosine pre-transfer RNA fragments are linked to p53-dependent neuronal cell death via PKM2.

Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, have been discovered to play an essential role in the pathogenesis of various disorders such as neurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutation of the CLP1 gene results in the accumulation of tRNA fragments of several different kinds. These tRNA fragments are expected to be associated with the disease pathogenesis. However, it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has the greatest impact on the onset of neuronal disease. We found that 5' tRNA fragments derived from tyrosine pre-tRNA (5' Tyr-tRF) caused p53-dependent neuronal cell death predominantly more than other types of tRNA fragment. We also showed that 5' Tyr-tRF bound directly to pyruvate kinase M2 (PKM2). Injection of zebrafish embryos with PKM2 mRNA ameliorated the neuronal defects induced in zebrafish embryos by 5' Tyr-tRF. Our findings partially uncovered a mechanistic link between 5' Tyr-tRF and neuronal cell death that is regulated by PKM2.

CLP1 acts as the main RNA kinase in mice.

CLP1 plays an essential role in the protein complex involved in mRNA 3'-end formation and polyadenylation as well as in the tRNA splicing endonuclease (TSEN) complex involved in the splicing of precursor tRNAs. NOL9 localizes in the nucleolus of cells and plays an essential role in ribosomal RNA maturation. Both CLP1 and NOL9 are RNA kinases that phosphorylate the 5' end of RNAs. From the evidence that phosphorylation of the 5' end of a siRNA is essential for its efficient RNA cleavage, it was expected that CLP1 and NOL9 would be corresponding molecules. However, there had been no direct evidence that this is the case. In this study, murine NOL9 showed no apparent RNA kinase activity in cells or even in an RNA kinase assay using recombinant murine NOL9 protein. Although siRNA efficiency was decreased in CLP1 kinase-dead (Clp1K/K) cells, it was not influenced by NOL9 overexpression. These findings indicate that in mouse cells it is CLP1 that mainly acts to phosphorylate the 5' end of RNAs in the siRNA pathway, with no apparent involvement of NOL9.

Induction of Airway Allergic Inflammation by Hypothiocyanite via Epithelial Cells.

Hypothiocyanite (OSCN-) serves as a potent innate defense system against microbes in the lungs. OSCN- is generated by the catalysis of peroxidases using thiocyanate transported via several anion transporters, including pendrin/SLC26A4 and hydrogen peroxide (H2O2) generated by Duox1 and Duox2. We previously demonstrated that expression of pendrin, peroxidases, and Duox1/Duox2 is up-regulated in bronchial asthma patients and/or asthma model mice and that these molecules are important in accelerating airway inflammation. However, it remained unclear how activating these molecules would lead to airway inflammation. In this study, we examined whether OSCN- produced via the pendrin/peroxidase/Duox pathway causes inflammation via airway epithelial cells. In an in vitro OSCN- production system, OSCN-, but not H2O2, activated NF-κB, a transcription factor critical for inflammatory responses, in the airway epithelial cells. OSCN- was sensed by protein kinase A (PKA) followed by formation of the dimerization of PKA. The dimerized PKA, the active form, was critical in activating NF-κB. Detoxifying H2O2, mainly by catalase, enabled the dominant abilities of OSCN- to dimerize PKA and activate NF-κB, compared with untreated H2O2 Furthermore, OSCN- in high doses caused necrosis of the cells, inducing release of IL-33, a trigger to initiate type 2 inflammation. These results demonstrate that OSCN- in low doses activates NF-κB via PKA in airway epithelial cells, whereas OSCN- in high doses causes necrosis, suggesting an important role in airway allergic inflammation for the production of OSCN- via the pendrin/peroxidase/Duox pathway.

Periostin contributes to epidermal hyperplasia in psoriasis common to atopic dermatitis.

BACKGROUND: Epidermal hyperplasia is a histological hallmark observed in both atopic dermatitis (AD) and psoriasis, although the clinical features and the underlying immunological disorders of these diseases are different. We previously showed that periostin, a matricellular protein, plays a critical role in epidermal hyperplasia in AD, using a mouse model and a 3-dimensional organotypic coculture system. In this study, we explore the hypothesis that periostin is involved in epidermal hyperplasia in psoriasis. METHODS: To examine expression of periostin in psoriasis patients, we performed immunohistochemical analysis on skin biopsies from six such patients. To investigate periostin's role in the pathogenesis of psoriasis, we evaluated periostin-deficient mice in a psoriasis mouse model induced by topical treatment with imiquimod (IMQ). RESULTS: Periostin was substantially expressed in the dermis of all investigated psoriasis patients. Epidermal hyperplasia induced by IMQ treatment was impaired in periostin-deficient mice, along with decreased skin swelling. However, upon treatment with IMQ, periostin deficiency did not alter infiltration of inflammatory cells such as neutrophils; production of IL-17, -22, or -23; or induction/expansion of IL-17- and IL-22-producing group 3 innate lymphoid cells. CONCLUSIONS: Periostin plays an important role during epidermal hyperplasia in IMQ-induced skin inflammation, independently of the IL-23-IL-17/IL-22 axis. Periostin appears to be a mediator for epidermal hyperplasia that is common to AD and psoriasis.

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.

[Establishment of novel biomarkers for Personalized medication for atopic dermatitis].

To diagnose atopic dermatitis (AD), an appearance of eczema examined by experienced dermatologists is required. Therefore, biomarkers to diagnose AD or to reflect the severity of AD would be of a great use for non-specialists in the clinic or hospitals. We can apply such a biomarker for realization of personalized medicine for AD in the future. Interleukin-4 (IL-4) and IL-13 have been known to play important roles in the pathogenesis of allergic diseases including AD. In addition to these, we previously identified SCCA1, SCCA2, and periostin as IL-4/IL-13-inducible genes. We recently established ELISA systems to measure serum levels of SCCA1, SCCA2, and periostin and evaluated their usefulness in the treatment of AD patients. Serum SCCA1 and SCCA2 are up-regulated in AD patients and can distinguish AD patients from non-atopic controls, and their serum levels reflect eczema grades. Periostin concentration is also elevated in the serum of AD patients. These results demonstrate that SCCA1, SCCA2, and periostin might be promising biomarkers for personalized medicine in allergic diseases including AD.

Zebrafish as a Useful Model System for Human Liver Disease.

Liver diseases represent a significant global health challenge, thereby necessitating extensive research to understand their intricate complexities and to develop effective treatments. In this context, zebrafish (Danio rerio) have emerged as a valuable model organism for studying various aspects of liver disease. The zebrafish liver has striking similarities to the human liver in terms of structure, function, and regenerative capacity. Researchers have successfully induced liver damage in zebrafish using chemical toxins, genetic manipulation, and other methods, thereby allowing the study of disease mechanisms and the progression of liver disease. Zebrafish embryos or larvae, with their transparency and rapid development, provide a unique opportunity for high-throughput drug screening and the identification of potential therapeutics. This review highlights how research on zebrafish has provided valuable insights into the pathological mechanisms of human liver disease.

Periostin, a matricellular protein, plays a role in the induction of chemokines in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually fatal form of interstitial lung disease (ILD). The precise molecular mechanisms of IPF remain poorly understood. However, analyses of mice receiving bleomycin (BLM) as a model of IPF established the importance of preceding inflammation for the formation of fibrosis. Periostin is a recently characterized matricellular protein involved in modulating cell functions. We recently found that periostin is highly expressed in the lung tissue of patients with IPF, suggesting that it may play a role in the process of pulmonary fibrosis. To explore this possibility, we administered BLM to periostin-deficient mice, and they subsequently showed a reduction of pulmonary fibrosis. We next determined whether this result was caused by a decrease in the preceding recruitment of neutrophils and macrophages in the lungs because of the lower production of chemokines and proinflammatory cytokines. We performed an in vitro analysis of chemokine production in lung fibroblasts, which indicated that periostin-deficient fibroblasts produced few or no chemokines in response to TNF-α compared with control samples, at least partly explaining the lack of inflammatory response and, therefore, fibrosis after BLM administration to periostin-deficient mice. In addition, we confirmed that periostin is highly expressed in the lung tissue of chemotherapeutic-agent-induced ILD as well as of patients with IPF. Taking these results together, we conclude that periostin plays a unique role as an inducer of chemokines to recruit neutrophils and macrophages important in the process of pulmonary fibrosis in BLM-administered model mice. Our results suggest a therapeutic potential for periostin in IPF and drug-induced ILD.

Forced expression of stabilized c-Fos in dendritic cells reduces cytokine production and immune responses in vivo.

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production by monocytic cells. We have shown that the transcription factor c-Fos is responsible for cAMP-mediated suppression of inflammatory cytokine production, and that c-Fos protein is stabilized by IKKß-mediated phosphorylation. We found that S308 is one of the major phosphorylation sites, and that the S308D mutation prolongs c-Fos halflife. To investigate the role of stabilized c-Fos protein in dendritic cells (DCs) in vivo, we generated CD11c-promoter-deriven c-FosS308D transgenic mice. As expected, bone marrow-derived DCs (BMDCs) from these Tg mice produced smaller amounts of inflammatory cytokines, including TNF-α, IL-12, and IL-23, but higher levels of IL-10, in response to LPS, than those from wild-type (Wt) mice. When T cells were co-cultured with BMDCs from Tg mice, production of Th1 and Th17 cytokines was reduced, although T cell proliferation was not affected. Tg mice demonstrated more resistance to experimental autoimmune encephalomyelitis (EAE) than did Wt mice. These data suggest that c-Fos in DCs plays a suppressive role in certain innate and adaptive immune responses.