RESUMEN
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.
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Autofagia , Hígado Graso , Hígado , Animales , Humanos , Autofagia/genética , Modelos Animales de Enfermedad , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado/metabolismo , Hígado/patología , Pez CebraRESUMEN
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.
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Envejecimiento Prematuro , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Envejecimiento Prematuro/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/deficiencia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Oxidativo , MutaciónRESUMEN
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.
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Hígado Graso , Neoplasias Hepáticas , Enfermedades Metabólicas , Perciformes , Animales , Pez Cebra , Adenosina , Cirrosis Hepática , Progresión de la Enfermedad , Adenosina TrifosfatoRESUMEN
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.
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Axones , Colesterol , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Pez Cebra , Animales , Autofagia , Axones/metabolismo , Colesterol/metabolismo , Lisosomas/metabolismo , Neurogénesis , Neuronas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/deficiencia , Síndrome de Smith-Lemli-Opitz/metabolismo , Síndrome de Smith-Lemli-Opitz/genética , Síndrome de Smith-Lemli-Opitz/patología , Pez Cebra/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
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Anticonvulsivantes , Epilepsia , Animales , Anticonvulsivantes/efectos adversos , Pez Cebra , Pentilenotetrazol/toxicidad , Ácido Glutámico , Serotonina , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Carbamazepina/farmacología , Levetiracetam/farmacología , Levetiracetam/uso terapéutico , Ácido gamma-Aminobutírico , NeurotransmisoresRESUMEN
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.
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Acidosis Tubular Renal , Acidosis , Pérdida Auditiva Sensorineural , Compuestos Organometálicos , ATPasas de Translocación de Protón Vacuolares , Animales , Humanos , Pez Cebra/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/patología , Mutación , Acidosis Tubular Renal/genética , Células Ciliadas Auditivas/patología , Concentración de Iones de Hidrógeno , Cabello/metabolismo , Adenosina TrifosfatoRESUMEN
Fibromyalgia (FM) is a syndrome characterized by chronic pain with depression as a frequent comorbidity. However, efficient management of the pain and depressive symptoms of FM is lacking. Given that endogenous oxytocin (OXT) contributes to the regulation of pain and depressive disorders, herein, we investigated the role of OXT in an experimental reserpine-induced FM model. In FM model, OXT-monomeric red fluorescent protein 1 (OXT-mRFP1) transgenic rats exhibited increased depressive behavior and sensitivity in a mechanical nociceptive test, suggesting reduced pain tolerance. Additionally, the development of the FM-like phenotype in OXT-mRFP1 FM model rats was accompanied by a significant reduction in OXT mRNA expression in the magnocellular neurons of the paraventricular nucleus. OXT-mRFP1 FM model rats also had significantly fewer tryptophan hydroxylase (TPH)- and tyrosine hydroxylase (TH)-immunoreactive (ir) neurons as well as reduced serotonin and norepinephrine levels in the dorsal raphe and locus coeruleus. To investigate the effects of stimulating the endogenous OXT pathway, rats expressing OXT-human muscarinic acetylcholine receptor (hM3Dq)-mCherry designer receptors exclusively activated by designer drugs (DREADDs) were also assessed in the FM model. Treatment of these rats with clozapine-N-oxide (CNO), an hM3Dq-activating drug, significantly improved characteristic FM model-induced pathophysiological pain, but did not alter depressive-like behavior. The chemogenetically induced effects were reversed by pre-treatment with an OXT receptor antagonist, confirming the specificity of action via the OXT pathway. These results indicate that endogenous OXT may have analgesic effects in FM, and could be a potential target for effective pain management strategies for this disorder.
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Fibromialgia , Oxitocina , Ratas , Humanos , Animales , Oxitocina/farmacología , Oxitocina/metabolismo , Reserpina/farmacología , Reserpina/metabolismo , Fibromialgia/inducido químicamente , Fibromialgia/metabolismo , Proteínas Luminiscentes/genética , Dolor/metabolismo , Ratas Transgénicas , Neuronas/metabolismo , Receptores de Oxitocina/metabolismoRESUMEN
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.
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Microcefalia , Pez Cebra , Animales , Péptidos y Proteínas de Señalización Intracelular , Microcefalia/genética , Proteínas Serina-Treonina Quinasas/genética , Pez Cebra/genéticaRESUMEN
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.
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Microcefalia , Infección por el Virus Zika , Virus Zika , Animales , Péptidos y Proteínas de Señalización Intracelular , Masculino , Microcefalia/genética , Microcefalia/patología , Mutación , Proteínas Serina-Treonina Quinasas , Espermatogénesis , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is properly entrained by environmental light cycle. However, the molecular mechanism(s) determining the magnitude of phase shift by light is still not fully understood. The orphan G-protein-coupled receptor Gpr176 is enriched in the SCN, controls the pace (period) of the circadian rhythm in behavior but is not apparently involved in the light entrainment; Gpr176-/- animals display a shortened circadian period in constant darkness but their phase-resetting responses to light are normal. Here, we performed microarray analysis and identified enhanced mRNA expression of neuromedin U (Nmu) and neuromedin S (Nms) in the SCN of Gpr176-/- mice. By generating C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice, we noted that the mutant mice had a greater magnitude of phase shift in response to early subjective night light than wildtype mice, while Nmu/Nms double knockout mice as well as Gpr176 knockout mice are normal in the phase shifts induced by light. At the molecular level, Nmu-/-Nms-/-Gpr176-/- mice had a reduced induction of Per1 and cFos mRNA expression in the SCN by light and mildly upregulated circadian expression of Per2, Prok2, Rgs16, and Rasl11b. These expressional changes may underlie the phenotype of the Nmu/Nms/Gpr176 knockout mice. Our data argue that there is a mechanism requiring Nmu, Nms, and Gpr176 for the proper modulation of light-induced phase shift in mice. Simultaneous modulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.
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Relojes Circadianos , Neuropéptidos , Núcleo Supraquiasmático , Animales , Relojes Circadianos/genética , Ritmo Circadiano/genética , Locomoción , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuropéptidos/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMEN
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.
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Estudio de Asociación del Genoma Completo , Proteínas Serina-Treonina Quinasas , Pez Cebra , Agresión , Animales , Femenino , Proteínas Serina-Treonina Quinasas/genética , Pez Cebra/genética , Ácido gamma-AminobutíricoRESUMEN
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.
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Enfermedades Cerebelosas/genética , Modelos Biológicos , Mutación/genética , Proteínas Nucleares/genética , Fosfotransferasas/genética , Precursores del ARN/metabolismo , ARN de Transferencia/metabolismo , Factores de Transcripción/genética , Tirosina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Enfermedades Cerebelosas/complicaciones , Fibroblastos/metabolismo , Humanos , Intrones/genética , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Microcefalia/complicaciones , Actividad Motora , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Proteínas Nucleares/química , Fenotipo , Fosfotransferasas/química , Factores de Transcripción/químicaRESUMEN
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.
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Anemia/patología , Autofagia , Leucina-ARNt Ligasa/deficiencia , Leucina/metabolismo , Fallo Hepático/patología , Pez Cebra/crecimiento & desarrollo , Anemia/enzimología , Anemia/etiología , Animales , Fallo Hepático/enzimología , Fallo Hepático/etiología , Pez Cebra/metabolismoRESUMEN
Obesity is now a public health concern. The leading cause of obesity is an energy imbalance between ingested and expended calories. The mechanisms of feeding behavior and energy metabolism are regulated by a complex of various kinds of molecules, including anorexigenic and orexigenic neuropeptides. One of these neuropeptides, neuromedin U (NMU), was isolated in the 1980s, and its specific receptors, NMUR1 and NMUR2, were defined in 2000. A series of subsequent studies has revealed many of the physiological roles of the NMU system, including in feeding behavior, energy expenditure, stress responses, circadian rhythmicity, and inflammation. Particularly over the past decades, many reports have indicated that the NMU system plays an essential and direct role in regulating body weight, feeding behavior, energy metabolism, and insulin secretion, which are tightly linked to obesity pathophysiology. Furthermore, another ligand of NMU receptors, NMS (neuromedin S), was identified in 2005. NMS has physiological functions similar to those of NMU. This review summarizes recent observations of the NMU system in relation to the pathophysiology of obesity in both the central nervous systems and the peripheral tissues.
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Neuropéptidos/metabolismo , Animales , Metabolismo Energético/fisiología , Conducta Alimentaria/fisiología , Humanos , Obesidad/metabolismoRESUMEN
The receptor-activator of NF-κB ligand (RANKL) and its specific receptor RANK have essential roles in regulating bone metabolism and the immune system. Besides, the RANKL/RANK system plays important roles in multiple physiological and pathophysiological processes such as mammary gland development during pregnancy, cancer development, and bone metastasis. While it has long been known that RANKL and RANK are expressed in the central nervous system (CNS), the physiological roles of RANKL/RANK system in the CNS and the underlying molecular mechanisms have been elucidated recently. Over the last decade, several reports showed that the central RANKL/RANK system plays important roles in regulating body temperature, brain ischemia, autoimmune encephalopathy, feeding behavior, and energy metabolism. In this review, it is provided an updated information regarding the roles of RANKL/RANK system in the CNS.
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Sistema Nervioso Central/metabolismo , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Animales , Temperatura Corporal , Sistema Nervioso Central/patología , Conducta Alimentaria , HumanosRESUMEN
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.
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Nucleótidos/metabolismo , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Embrión no Mamífero/anomalías , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Larva/genética , Larva/fisiología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Proteína Básica de Mielina/genética , Nucleótidos/genética , Sirolimus/farmacología , Pez Cebra/embriologíaRESUMEN
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.
RESUMEN
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.
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Neuronas/enzimología , Neuronas/patología , Piruvato Quinasa/metabolismo , Precursores del ARN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Tirosina/metabolismo , Animales , Muerte Celular , Diferenciación Celular , Línea Celular , Embrión no Mamífero/metabolismo , Humanos , Pez Cebra/embriologíaRESUMEN
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.