ABSTRACT
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.
Subject(s)
Autophagy , Fatty Liver , Liver , Zebrafish , Animals , Autophagy/genetics , Fatty Liver/genetics , Fatty Liver/metabolism , Fatty Liver/pathology , Liver/metabolism , Liver/pathology , Humans , Disease Models, AnimalABSTRACT
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.
Subject(s)
Aging, Premature , Nonsense Mediated mRNA Decay , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/genetics , Aging, Premature/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Zebrafish Proteins/deficiency , RNA, Messenger/genetics , RNA, Messenger/metabolism , Oxidative Stress , MutationABSTRACT
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.
Subject(s)
Axons , Cholesterol , Oxidoreductases Acting on CH-CH Group Donors , Zebrafish , Animals , Autophagy , Axons/metabolism , Cholesterol/metabolism , Lysosomes/metabolism , Neurogenesis , Neurons/metabolism , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Oxidoreductases Acting on CH-CH Group Donors/genetics , Oxidoreductases Acting on CH-CH Group Donors/deficiency , Smith-Lemli-Opitz Syndrome/metabolism , Smith-Lemli-Opitz Syndrome/genetics , Smith-Lemli-Opitz Syndrome/pathology , Zebrafish/metabolism , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Steatotic liver disease (SLD) is a burgeoning health problem predominantly associated with excessive alcohol consumption, which causes alcohol-related liver disease (ALD), and high caloric intake, which results in metabolic dysfunction-associated SLD (MASLD). The pathogenesis of ALD and MASLD, which can progress from steatohepatitis to more severe conditions such as liver fibrosis, cirrhosis, and hepatocellular carcinoma, is complicated by several factors. Recently, extracellular ATP and adenosine (Ado), as damage-associated molecular patterns, were reported to promote inflammation and liver fibrosis, contributing to SLD pathogenesis. Here, we explored the in vivo dynamics of hepatic extracellular ATP and Ado during the progression of steatohepatitis using a genetically encoded GPCR-activation-based sensor (GRAB) in zebrafish models. We established hepatocyte-specific GRABATP and GRABAdo in zebrafish and investigated the changes in in vivo hepatic extracellular ATP and Ado levels under ALD or MASLD conditions. Disease-specific changes in hepatocyte extracellular ATP and Ado levels were observed, clearly indicating a correlation between hepatocyte extracellular ATP/Ado dynamics and disease progression. Furthermore, clodronate, a vesicular nucleotide transporter inhibitor, alleviated the MASLD phenotype by reducing the hepatic extracellular ATP and Ado content. These findings provide deep insights into extracellular ATP/Ado dynamics in disease progression, suggesting therapeutic potential for ALD and MASLD.
Subject(s)
Fatty Liver , Liver Neoplasms , Metabolic Diseases , Perciformes , Animals , Zebrafish , Adenosine , Liver Cirrhosis , Disease Progression , Adenosine TriphosphateABSTRACT
Epilepsy, a recurrent neurological disorder involving abnormal neurotransmitter kinetics in the brain, has emerged as a global health concern. The mechanism of epileptic seizures is thought to involve a relative imbalance between excitatory and inhibitory neurotransmitters. Despite the recent advances in clinical and basic research on the pathogenesis of epilepsy, the complex relationship between the neurotransmitter changes and behavior with and without antiepileptic drugs (AEDs) during seizures remains unclear. To investigate the effects of AEDs such as levetiracetam (LEV), carbamazepine (CBZ), and fenfluramine (FFR) on key neurotransmitters in the pentylenetetrazol (PTZ)-induced seizures in adult zebrafish, we examined the changes in glutamic acid, gamma-aminobutyric acid (GABA), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), choline, acetylcholine, norepinephrine, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and adenosine. In this study, we observed that 5-HT and DA levels in the brain increased immediately after PTZ-induced seizures. Behavioral tests clearly showed that all of these AEDs suppressed the PTZ-induced seizures. Upon treatment of PTZ-induced seizures with these AEDs, CBZ decreased the glutamic acid and FFR increased the GABA levels; however, no neurotransmitter changes were observed in the brain after LEV administration. Thus, we demonstrated a series of neurotransmitter changes linked to behavioral changes during PTZ-induced epileptic seizures when LEV, CBZ, or FFR were administered. These findings will lead to a more detailed understanding of the pathogenesis of epilepsy associated with behavioral and neurotransmitter changes under AED treatment.
Subject(s)
Anticonvulsants , Epilepsy , Animals , Anticonvulsants/adverse effects , Zebrafish , Pentylenetetrazole/toxicity , Glutamic Acid , Serotonin , Seizures/chemically induced , Seizures/drug therapy , Carbamazepine/pharmacology , Levetiracetam/pharmacology , Levetiracetam/therapeutic use , gamma-Aminobutyric Acid , Neurotransmitter AgentsABSTRACT
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.
Subject(s)
Aortic Aneurysm, Thoracic , Heart Defects, Congenital , Animals , Humans , Actins/genetics , Actins/metabolism , Zebrafish/metabolism , Mutation, Missense , Aortic Aneurysm, Thoracic/geneticsABSTRACT
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.
Subject(s)
Acidosis, Renal Tubular , Acidosis , Hearing Loss, Sensorineural , Organometallic Compounds , Vacuolar Proton-Translocating ATPases , Animals , Humans , Zebrafish/metabolism , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolism , Hearing Loss, Sensorineural/genetics , Hearing Loss, Sensorineural/pathology , Mutation , Acidosis, Renal Tubular/genetics , Hair Cells, Auditory/pathology , Hydrogen-Ion Concentration , Hair/metabolism , Adenosine TriphosphateABSTRACT
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.
Subject(s)
Liver Diseases , Zebrafish , Humans , Animals , High-Throughput Screening Assays , LarvaABSTRACT
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.
Subject(s)
Microcephaly , Zebrafish , Animals , Intracellular Signaling Peptides and Proteins , Microcephaly/genetics , Protein Serine-Threonine Kinases/genetics , Zebrafish/geneticsABSTRACT
Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway is essential for tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). To date, however, almost all clinical trials of inhibitor targeting this pathway have failed to improve the outcome of patients with PDAC. We found that implanted MIA Paca2, a human PDAC cell line sensitive to a MAPK inhibitor, PD0325901, became refractory within a week after treatment. By comparing the expression profiles of MIA Paca2 before and after acquisition of the refractoriness to PD0325901, we identified clusterin (CLU) as a candidate gene involved. CLU was shown to be induced immediately after treatment with PD0325901 or expressed primarily in more than half of PDAC cell lines, enhancing cell viability by escaping from apoptosis. A combination of PD0325901 and CLU downregulation was found to synergistically or additively reduce the proliferation of PDAC cells. In surgically resected PDAC tissues, overexpression of CLU in cancer cells was observed immunohistochemically in approximately half of the cases studied. Collectively, our findings highlight the mechanisms responsible for the rapid refractory response to MEK inhibitor in PDAC cells, suggesting a novel therapeutic strategy that could be applicable to patients with PDAC using inhibitor targeting the MAPK signaling pathway and CLU.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Clusterin/genetics , Clusterin/metabolism , Clusterin/therapeutic use , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase Kinases , Cell Line, Tumor , Cell Proliferation , Pancreatic NeoplasmsABSTRACT
Despite recent advances in sequencing technology and large-scale drug screenings employing hundreds of cell lines, the predictive accuracy of mutation-based biomarkers is still insufficient as a guide for cancer therapy. Therefore, novel types of diagnostic methods using alternative biomarkers would be highly desirable. We have hypothesized that sensitivity-specific changes in the phosphorylation of signaling molecules could be useful in this respect. Here, with the aim of developing a method for predicting the response of cancers to cisplatin using a combination of specific biomarker(s) and patient-derived tumor organoids (PDOs), we found that cisplatin-sensitive cell lines or PDOs showed enhanced phosphorylation of c-Jun (p-c-Jun) within 24 h after cisplatin treatment. We also compared the responses of 6 PDOs to cisplatin with the therapeutic effect of neoadjuvant chemotherapy (docetaxel/cisplatin/5-fluorouracil) in 6 matched patients. Mechanistically, the c-Jun induction was partly related to TNF signaling induced by cisplatin. Our data suggest that enhanced phosphorylation of c-Jun in response to cisplatin treatment could be a predictive biomarker for the efficacy of cisplatin in selected cancer patients.
Subject(s)
Antineoplastic Agents , Neoplasms , Humans , Cisplatin/pharmacology , Cisplatin/therapeutic use , Organoids/pathology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Phosphorylation , Docetaxel/pharmacology , Neoplasms/pathology , BiomarkersABSTRACT
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.
Subject(s)
Microcephaly , Zika Virus Infection , Zika Virus , Animals , Intracellular Signaling Peptides and Proteins , Male , Microcephaly/genetics , Microcephaly/pathology , Mutation , Protein Serine-Threonine Kinases , Spermatogenesis , Zebrafish/genetics , Zebrafish/metabolismABSTRACT
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.
Subject(s)
Genome-Wide Association Study , Protein Serine-Threonine Kinases , Zebrafish , Aggression , Animals , Female , Protein Serine-Threonine Kinases/genetics , Zebrafish/genetics , gamma-Aminobutyric AcidABSTRACT
The liver function is essential for metabolism, detoxification, and bile synthesis, even in the neonatal period. Autophagy plays significance roles in THE adult liver, whereas the role of liver autophagy in the early neonatal period largely remains unclear. To clarify the importance of liver autophagy in the neonatal starvation period, we generated liver-specific autophagy-deficient (Atg5flox/flox; Albumin-Cre) mice and investigated under starvation conditions comparing with control (Atg5flox/+; Albumin-Cre) mice, focusing on serum metabolites and liver histopathology. As a result, autophagy in the liver was found to unessential for the survival under postnatal starvation. A metabolomics analysis of serum metabolites by gas chromatography-tandem mass spectrometry showed a significant difference between the groups, especially after 12-h starvation, suggesting the synergistical adaption of metabolic pathways, such as the "malate-aspartate shuttle", "aspartate metabolism", "urea cycle", and "glycine and serine metabolism". Liver-specific autophagy-deficiency under postnatal starvation conditions can cause a characteristic metabolic alteration suggesting a change of the mitochondrial function. Neonates seemed to maintain ketone production under starvation conditions, even in the autophagy-deficient liver, through a change in the mitochondrial function, which may be an adaptive mechanism for avoiding fatal starvation.
Subject(s)
Autophagy-Related Protein 5/deficiency , Autophagy , Liver/metabolism , Metabolome , Starvation/blood , Animals , Animals, Newborn , Gas Chromatography-Mass Spectrometry , MiceABSTRACT
The zebrafish is a valuable model organism that is widely used in studies of vertebrate development. In the laboratory, zebrafish embryonic development is normally carried out at 28.5°C. In this study, we sought to determine whether it was possible to modify the speed of embryonic development through the use of short- and long-term variations in incubation temperature. After incubation at 20°C-32°C, most early-stage embryos survived to the epiboly stage, whereas more than half of the embryos died at <20°C or >32°C. The rate of development differed between embryos incubated at the lowest (18°C) and highest (34°C) temperatures: a difference of 60 min was observed at the 2-cell stage and 290 min at the 1k-cell stage. When blastulae that had developed at 28°C were transferred to a temperature lower than 18°C for one or more hours, they developed normally after being returned to the original 28°C. Analyses using green fluorescent protein-buckyball mRNA and in situ hybridization against vasa mRNA showed that primordial germ cells increase under low-temperature culture; this response may be of use for studies involving heterochronic germ cell transplantation. Our study shows that embryonic developmental speed can be slowed, which will be of value for performing time-consuming, complicated, and delicate microsurgical operations.
Subject(s)
Embryo, Nonmammalian , Zebrafish , Animals , Blastula , Embryonic Development , TemperatureABSTRACT
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.
Subject(s)
Cerebellar Diseases/genetics , Models, Biological , Mutation/genetics , Nuclear Proteins/genetics , Phosphotransferases/genetics , RNA Precursors/metabolism , RNA, Transfer/metabolism , Transcription Factors/genetics , Tyrosine/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cerebellar Diseases/complications , Fibroblasts/metabolism , Humans , Introns/genetics , Mice, Inbred C57BL , Mice, Inbred ICR , Microcephaly/complications , Motor Activity , Motor Neurons/metabolism , Motor Neurons/pathology , Nuclear Proteins/chemistry , Phenotype , Phosphotransferases/chemistry , Transcription Factors/chemistryABSTRACT
Leucyl-tRNA synthetase (LARS) is an enzyme that catalyses the ligation of leucine with leucine tRNA. LARS is also essential to sensitize the intracellular leucine concentration to the mammalian target of rapamycin complex 1 (mTORC1) activation. Biallelic mutation in the LARS gene causes infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute liver failure, anaemia, and neurological disorders, including microcephaly and seizures. However, the molecular mechanism underlying ILFS1 under LARS deficiency has been elusive. Here, we generated Lars deficient (larsb-/-) zebrafish that showed progressive liver failure and anaemia, resulting in early lethality within 12 days post fertilization. The atg5-morpholino knockdown and bafilomycin treatment partially improved the size of the liver and survival rate in larsb-/- zebrafish. These findings indicate the involvement of autophagy in the pathogenesis of larsb-/- zebrafish. Indeed, excessive autophagy activation was observed in larsb-/- zebrafish. Therefore, our data clarify a mechanistic link between LARS and autophagy in vivo. Furthermore, autophagy regulation by LARS could lead to development of new therapeutics for IFLS1.
Subject(s)
Anemia/pathology , Autophagy , Leucine-tRNA Ligase/deficiency , Leucine/metabolism , Liver Failure/pathology , Zebrafish/growth & development , Anemia/enzymology , Anemia/etiology , Animals , Liver Failure/enzymology , Liver Failure/etiology , Zebrafish/metabolismABSTRACT
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.
Subject(s)
Nucleotides/metabolism , Zebrafish/genetics , Animals , Animals, Genetically Modified , CRISPR-Cas Systems , Embryo, Nonmammalian/abnormalities , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Larva/genetics , Larva/physiology , Motor Neurons/drug effects , Motor Neurons/pathology , Myelin Basic Protein/genetics , Nucleotides/genetics , Sirolimus/pharmacology , Zebrafish/embryologyABSTRACT
Neuromedin U (NMU) is a bioactive neuropeptide, highly distributed in the gastrointestinal tract and the central nervous system. NMU has various physiological functions related to feeding behavior, energy metabolism, stress responses, circadian rhythmicity and inflammation. Recently, several reports indicate that the central NMU system plays an important role in the reward systems in the brain. However, the underlying molecular mechanisms are not yet fully defined. In this study, we found that some of cocaine-induced c-Fos immunoreactive cells were co-localized with NMU in the nucleus accumbens (NAc), caudate putamen (CPu), and basolateral amygdala (BLA), which are key brain regions associated with the brain reward system, in wild type mice. Whereas, a treatment with cocaine did not influence the kinetics of NMU or NMU receptors mRNA expression in these brain regions, and NMU-knockout mice did not show any higher preference for cocaine compared with their control mice. These results indicate that cocaine has some effect on NMU expressing neurons related to the brain reward system, and this suggests NMU system may have a role on the brain reward systems activated by cocaine.