RESUMEN
Autism Spectrum Disorders (ASD) comprise a range of early age-onset neurodevelopment disorders with genetic heterogeneity. Most ASD related genes are involved in synaptic function, which is regulated by mature brain-derived neurotrophic factor (mBDNF) and its precursor proBDNF in a diametrically opposite manner: proBDNF inhibits while mBDNF potentiates synapses. Here we generated a knock-in mouse line (BDNFmet/leu) in which the conversion of proBDNF to mBDNF is attenuated. Biochemical experiments revealed residual mBDNF but excessive proBDNF in the brain. Similar to other ASD mouse models, the BDNFmet/leu mice showed reduced dendritic arborization, altered spines, and impaired synaptic transmission and plasticity in the hippocampus. They also exhibited ASD-like phenotypes, including stereotypical behaviors and deficits in social interaction. Moreover, the plasma proBDNF/mBDNF ratio was significantly increased in ASD patients compared to normal children in a case-control study. Thus, deficits in proBDNF to mBDNF conversion in the brain may contribute to ASD-like behaviors, and plasma proBDNF/mBDNF ratio may be a potential biomarker for ASD.
RESUMEN
While brain-derived neurotrophic factor (BDNF), which is a growth factor associated with cognitive improvement and the alleviation of depression symptoms, is known to regulate food intake and body weight, the role of BDNF in peripheral disease is not fully understood. Here, we show that reduced BDNF expression is associated with weight gain and the chronic liver disease non-alcoholic steatohepatitis (NASH). At 10 months of age, BDNF-heterozygous (BDNF+/- ) mice developed symptoms of NASH: centrilobular/perivenular steatosis, lobular inflammation with infiltration of neutrophils, ballooning hepatocytes, and fibrosis of the liver. Obesity and higher serum levels of glucose and insulin - major pathologic features in human NASH - were dramatic. Dying adipocytes were surrounded by macrophages in visceral fat, suggesting that chronic inflammation occurs in peripheral organs. RNA sequencing (RNA-seq) studies of the liver revealed that the most significantly enriched Gene Ontology term involved fatty acid metabolic processes and the modulation of neutrophil aggregation, pathologies that well characterise NASH. Gene expression analysis by RNA-seq also support the notion that BDNF+/- mice are under oxidative stress, as indicated by alterations in the expression of the cytochrome P450 family and a reduction in glutathione S-transferase p, an antioxidant enzyme. Histopathologic phenotypes of NASH were also observed in a knock-in mouse (BDNF+/pro ), in which the precursor BDNF is inefficiently converted into the mature form of BDNF. Lastly, as BDNF reduction causes overeating and subsequent obesity, a food restriction study was conducted in BDNF+/pro mice. Pair-fed BDNF+/pro mice developed hepatocellular damage and showed infiltration of inflammatory cells, including neutrophils in the liver, despite having body weights and blood parameters that were comparable to those of controls. This is the first report demonstrating that reduced BDNF expression plays a role in the pathogenic mechanism of NASH, which is a hepatic manifestation of metabolic syndrome. © 2023 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Humanos , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Ratones Noqueados , Hígado/patología , Inflamación/patología , Obesidad/complicaciones , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Dieta Alta en GrasaRESUMEN
Brain-derived neurotrophic factor (BDNF) is known to control a wide variety of brain functions, ranging from memory formation to food intake. However, since the BDNF levels are extremely low in the nervous system, the dynamics in neurons from intracellular trafficking to secretion is absolutely complicated; the understanding is not fully promoted. We here review the findings of those critical mechanisms from intracellular trafficking to the secretion of BDNF. Furthermore, to solve this issue, technological advances for the detection, measurement, and imaging of this growth factor are essential. We believe that this review helps the study of these complex but critical mechanisms of BDNF.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Transporte de Proteínas/genética , Humanos , Transmisión SinápticaRESUMEN
We generated a knock-in mouse line in which the gene encoding brain-derived neurotrophic factor (Bdnf) was replaced with a sequence for proBDNF containing human single nucleotide polymorphisms encoding arginines proximal to the cleavage site (R125M and R127L). The ratio of the mature form of BDNF (mBDNF) to precursor BDNF (proBDNF) in hippocampal tissue lysates was decreased in a manner dependent on the number of copies of the mutant gene, indicating that the mutations inhibited proteolytic conversion of proBDNF into mBDNF. Although homozygous mice had a proBDNF/mBDNF ratio of ~9:1, they survived until adulthood. The levels of mBDNF were reduced by 57% in heterozygous mutant mice, which exhibited a depressive-like behavior in the tail suspension test and weight gain when housed in social isolation, showing that impaired proBDNF cleavage contributes to stress-induced depressive-like phenotypes. Furthermore, socially isolated heterozygous mice displayed a pronounced deficit in daily nest-building behaviors. These findings suggest that the decreased production of mBDNF by impaired proBDNF cleavage disturbs daily activities in mice.
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Conducta Animal , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Depresión/metabolismo , Alelos , Animales , Peso Corporal , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Genotipo , Heterocigoto , Hipocampo/metabolismo , Homocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Fenotipo , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Aislamiento SocialRESUMEN
Most growth factors are synthesized as precursors and biologically active forms are generated by proteolytic cleavage of the pro-domain. However, the biological functions of pro-domains are ill-defined. New roles were recently reported for the pro-domain of brain-derived neurotrophic factor (BDNF), a well-known growth factor in the brain. Interestingly, the pro-domain of BDNF (BDNF pro-peptide) is localized at presynaptic termini, where it facilitates long-term depression (LTD) in hippocampal slices, implicating it as a novel synaptic modulator. BDNF binds its pro-peptide with high affinity in a pH-dependent manner and when bound to BDNF, the BDNF pro-peptide cannot facilitate hippocampal LTD, representing a new mechanism of regulation. The BDNF pro-peptide is present in human cerebrospinal fluid (CSF) and levels were significantly lower in patients with major depressive disorder (MDD) than in controls. Notably, male MDD patients exhibit significantly lower levels of CSF pro-peptide than females. These findings demonstrate that the BDNF pro-peptide is a biologically important synaptic modulator and is associated with MDD, particularly in males.
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Factor Neurotrófico Derivado del Encéfalo/metabolismo , Trastorno Depresivo Mayor/metabolismo , Hipocampo/metabolismo , Depresión Sináptica a Largo Plazo/fisiología , Neurotransmisores/metabolismo , Terminales Presinápticos/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Ratones , Precursores de Proteínas/líquido cefalorraquídeo , Precursores de Proteínas/metabolismo , Ratas , Transmisión SinápticaRESUMEN
The role of brain-derived neurotrophic factor (BDNF) and its related molecules has been extensively studied in the context of psychiatric disorders. In the present study, we focused on the newly identified BDNF pro-peptide, which is generated together with mature BDNF by proteolytic processing of their precursor, proBDNF. Here, we report, for the first time, that BDNF pro-peptide is present in human cerebrospinal fluid (CSF) and quantifiable by western blotting. We measured CSF BDNF pro-peptide levels in 27 patients with schizophrenia, 18 patients with major depressive disorder (MDD), and 27 healthy controls matched for age, sex, and ethnicity (Japanese). The ratio of the BDNF pro-peptide level to the total protein level in MDD patients was significantly lower than that in controls (Kruskal-Wallis with Dunn's multiple comparisons test; pâ¯=â¯0.046). When men and women were examined separately, males with MDD had a significantly lower BDNF pro-peptide/protein ratio than male controls (pâ¯=â¯0.047); this difference was not found in female subjects. The ratio tended to be lower in male schizophrenia patients (pâ¯=â¯0.10). Although we tried to measure the levels of mature BDNF in CSF, they were below the limit of detection of the ELISA and multiple analyte profiling technology. Taken together, the results suggest that reduced CSF BDNF pro-peptide levels are associated with MDD, particularly in males. Further studies involving a larger sample size are warranted.
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Factor Neurotrófico Derivado del Encéfalo/líquido cefalorraquídeo , Trastorno Depresivo Mayor/líquido cefalorraquídeo , Esquizofrenia/líquido cefalorraquídeo , Adulto , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Synaptic plasticity underlies higher brain function such as learning and memory, and the actin cytoskeleton in dendritic spines composing excitatory postsynaptic sites plays a pivotal role in synaptic plasticity. In this chapter, we review the role of drebrin in the regulation of the actin cytoskeleton during synaptic plasticity, under long-term potentiation (LTP) and long-term depression (LTD). Dendritic spines have two F-actin pools, drebrin-decorated stable F-actin (DF-actin) and drebrin-free dynamic F-actin (FF-actin). Resting dendritic spines change their shape, but are fairly constant over time at steady state because of the presence of DF-actin. Accumulation of DF-actin is inversely regulated by the intracellular Ca2+ concentration. However, LTP and LTD stimulation induce Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors into the potentiated spines, resulting in drebrin exodus via myosin II ATPase activation. The potentiated spines change to excited state because of the decrease in DF-actin and thus change their shape robustly. In LTP, the Ca2+ increase via NMDA receptors soon returns to the basal level, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression at the postsynaptic membrane is increased. The Ca2+ recovery and AMPAR increase coordinately induce the re-accumulation of DF-actin and change the dendritic spines from the excited state to steady state during LTP maintenance. During LTD, the prolonged intracellular Ca2+ increase inhibits the re-accumulation of DF-actin, resulting in facilitation of AMPAR endocytosis. Because of the positive feedback loop of the AMPAR decrease and drebrin re-accumulation inhibition, the dendritic spines are instable during LTD maintenance. Taken together, we propose the presence of resilient spines at steady state and plastic spines at excited state and discuss the physiological and pathological relevance of the two-state model to synaptic plasticity.
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Espinas Dendríticas/metabolismo , Plasticidad Neuronal/genética , Neuronas/metabolismo , Neuropéptidos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Espinas Dendríticas/genética , Neuropéptidos/genética , Sinapsis/metabolismo , Membranas Sinápticas/metabolismoRESUMEN
Most growth factors are initially synthesized as precursors and it was cleaved into bioactive mature domain and pro-domain. However, compared with the expression and function of bioactive mature domain, the biological role of the pro-domain is poorly understood. Unexpectedly, we found that the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which is well-known neurotrophic factor in brain, has a potential ability to facilitate hippocampal long-term depression. Furthermore, a BDNF polymorphism Val66Met, which substitute valine into methionine at 66 amino acid, impacted the biological activity of the BDNF pro-peptide. We lastly discuss the possible roles of BDNF and its pro-peptide in the generation of neural stem cells and progress of ischemia.
RESUMEN
Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Mutación Missense , Polimorfismo de Nucleótido Simple , Animales , Factor Neurotrófico Derivado del Encéfalo/química , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Multimerización de Proteína , ProteolisisRESUMEN
Brain-derived neurotrophic factor (BDNF) is one of the most active members of the neurotrophin family. BDNF not only regulates neuronal survival and differentiation, but also functions in activity-dependent plasticity processes such as long-term potentiation (LTP), long-term depression (LTD), learning, and memory. Like other growth factors, BDNF is produced by molecular and cellular mechanisms including transcription and translation, and functions as a bioactive molecule in the nervous system. Among these mechanisms, a particular post-translational mechanism, namely the conversion of precursor BDNF into mature BDNF by proteolytic cleavage, was not fully understood. In this review, we discuss the manner through which this post-translational mechanism alters the biological actions of BDNF protein. In addition to the initially elucidated findings on BDNF, the biological roles of precursor BDNF and the BDNF pro-peptide, especially synaptic plasticity, will be extensively discussed. Recent findings on the BDNF pro-peptide will provide new insights for understanding the mechanisms of action of the pro-peptides of growth factors.
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Factor Neurotrófico Derivado del Encéfalo/metabolismo , Plasticidad Neuronal , Precursores de Proteínas/metabolismo , Transducción de Señal , Sinapsis/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/análisis , Factor Neurotrófico Derivado del Encéfalo/genética , Humanos , Depresión Sináptica a Largo Plazo , Polimorfismo Genético , Precursores de Proteínas/análisis , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Transmisión SinápticaRESUMEN
Most growth factors are initially synthesized as precursor proteins and subsequently processed into their mature form by proteolytic cleavage, resulting in simultaneous removal of a pro-peptide. However, compared with that of mature form, the biological role of the pro-peptide is poorly understood. Here, we investigated the biological role of the pro-peptide of brain-derived neurotrophic factor (BDNF) and first showed that the pro-peptide is expressed and secreted in hippocampal tissues and cultures, respectively. Interestingly, we found that the BDNF pro-peptide directly facilitates hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA receptors and the pan-neurotrophin receptor p75(NTR). The BDNF pro-peptide also enhances NMDA-induced α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor endocytosis, a mechanism crucial for LTD expression. Thus, the BDNF pro-peptide is involved in synaptic plasticity that regulates a mechanism responsible for promoting LTD. The well-known BDNF polymorphism valine for methionine at amino acid position 66 (Val66Met) affects human memory function. Here, the BDNF pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings demonstrate functional roles for the BDNF pro-peptide and a naturally occurring human BDNF polymorphism in hippocampal synaptic depression.
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Factor Neurotrófico Derivado del Encéfalo/fisiología , Hipocampo/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Metionina/genética , Polimorfismo Genético , Precursores de Proteínas/fisiología , Valina/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Humanos , Ratones , Ratones Noqueados , Precursores de Proteínas/genética , RatasRESUMEN
Non-proliferating cells, such as mature neurons, are generally believed to be more resistant to X-rays than proliferating cells, such as glial and vascular endothelial cells. Therefore, the late adverse effects of radiotherapy on the brain have been attributed to the radiation-induced damage of glial and vascular endothelial cells. However, little is known about the radiosensitivities of neurons and glial cells due to difficulties in culturing these cells, particularly neurons, independently. In the present study, primary dissociated neurons and glial cultures were prepared separately from the hippocampi and cerebrum, respectively, which had been obtained from the same fetal rat on embryonic day 18. X-irradiations of 50 Gy were performed on the cultured neurons and glial cells at 7 and 21 days in vitro (DIV). The cells were fixed at 24 h after irradiation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was then performed to measure the apoptotic indices (AIs). The AIs of non-irradiated and irradiated neurons at 7 DIV were 23.7±6.7 and 64.9±4.8%, and those at 21 DIV were 52.1±17.4 and 44.6±12.5%, respectively. The AIs of non-irradiated and irradiated glial cells at 7 DIV were 5.8±1.5 and 78.4±3.3% and those at 21 DIV were 9.6±2.6 and 86.3±4.9%, respectively. Glial cells and neurons were radiosensitive at 7 DIV. However, while glial cells were radiosensitive at 21 DIV, neurons were not.
RESUMEN
The neuronal actin-binding protein drebrin A forms a stable structure with F-actin in dendritic spines. NMDA receptor activation causes an exodus of F-actin bound by drebrin A (DA-actin) from dendritic spines, suggesting a pivotal role for DA-actin exodus in synaptic plasticity. We quantitatively assessed the extent of DA-actin localization to spines using the spine-dendrite ratio of drebrin A in cultured hippocampal neurons, and found that (1) chemical long-term potentiation (LTP) stimulation induces rapid DA-actin exodus and subsequent DA-actin re-entry in dendritic spines, (2) Ca(2+) influx through NMDA receptors regulates the exodus and the basal accumulation of DA-actin, and (3) the DA-actin exodus is blocked by myosin II ATPase inhibitor, but is not blocked by myosin light chain kinase (MLCK) or Rho-associated kinase (ROCK) inhibitors. These results indicate that myosin II mediates the interaction between NMDA receptor activation and DA-actin exodus in LTP induction. Furthermore, myosin II seems to be activated by a rapid actin-linked mechanism rather than slow MLC phosphorylation. Thus the myosin-II mediated DA-actin exodus might be an initial event in LTP induction, triggering actin polymerization and spine enlargement.
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Actinas/metabolismo , Espinas Dendríticas/metabolismo , Potenciación a Largo Plazo/fisiología , Miosina Tipo II/metabolismo , Neuropéptidos/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Bicuculina/farmacología , Calcio/metabolismo , Células Cultivadas , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/fisiología , Femenino , Antagonistas de Receptores de GABA-A/farmacología , Ácido Glutámico/farmacología , Glicina/farmacología , Hipocampo/citología , Potenciación a Largo Plazo/efectos de los fármacos , Microscopía Fluorescente , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Embarazo , Unión Proteica , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Tetrodotoxina/farmacología , Imagen de Lapso de TiempoRESUMEN
Dendritic spines are small, actin-rich protrusions on dendrites, the development of which is fundamental for the formation of neural circuits. The actin cytoskeleton is central to dendritic spine morphogenesis. Drebrin is an actin-binding protein that is thought to initiate spine formation through a unique drebrin-actin complex at postsynaptic sites. However drebrin overexpression in neurons does not increase the final density of dendritic spines. In this study, we have identified and characterized a novel drebrin-binding protein, spikar. Spikar is localized in cell nuclei and dendritic spines, and accumulation of spikar in dendritic spines directly correlates with spine density. A reporter gene assay demonstrated that spikar acts as a transcriptional co-activator for nuclear receptors. We found that dendritic spine, but not nuclear, localization of spikar requires drebrin. RNA-interference knockdown and overexpression experiments demonstrated that extranuclear spikar regulates dendritic spine density by modulating de novo spine formation and retraction of existing spines. Unlike drebrin, spikar does not affect either the morphology or function of dendritic spines. These findings indicate that drebrin-mediated postsynaptic accumulation of spikar regulates spine density, but is not involved in regulation of spine morphology.
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Espinas Dendríticas/fisiología , Neuropéptidos/metabolismo , Transactivadores/fisiología , Animales , Western Blotting , Células Cultivadas , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/genética , Fenómenos Electrofisiológicos , Femenino , Genes Reporteros/genética , Vectores Genéticos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Embarazo , Interferencia de ARN , Ratas , Saccharomyces cerevisiae , Fracciones Subcelulares/metabolismo , Sinapsis/fisiología , TransfecciónRESUMEN
Neurons are essential components of neural circuits and provide brain function organization. We previously reported that X irradiation induces apoptosis in immature neurons. To the best of our knowledge, there have been few reports investigating the effects of X irradiation on mature neurons. We analyzed the effects of X irradiation on the morphology, density and cytoskeletal proteins in dendritic spines on mature neurons. We prepared developing hippocampal neurons from 18 days embryo by using Banker's method. Neurons at 21 days in vitro were X irradiated at several doses and were immediately fixed. To evaluate the dendritic spine morphology and density, the neurons were transfected with a reporter plasmid for enhanced green fluorescent protein (GFP). Changes in the dendritic spines as a result of X irradiation were evaluated using electron microscopy. To analyze the cytoskeletal proteins within the dendritic spines, we performed immunocytochemistry to detect filamentous actin (F-actin), drebrin and PSD-95. X irradiation immediately changed the dendritic spine morphology, and the irradiated spines were significantly thinner and longer than the nonirradiated spines. X irradiation decreased the dendritic spine density in a dose-dependent manner. Electron microscopy confirmed these changes of dendritic spines by X irradiation. Immunohistochemical studies showed that X irradiation decreased the accumulation of drebrin and F-actin, but not PSD-95, within the dendritic spines. These results suggest that X irradiation immediately decreases the dendritic spine density and changes the morphology of mature neurons by reducing the abundance of cytoskeletal proteins. The abnormal dendritic spines may be associated with acute adverse effects after X irradiation in a clinical setting, although further investigations are warranted to validate these findings.
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Proteínas del Citoesqueleto/metabolismo , Espinas Dendríticas/metabolismo , Espinas Dendríticas/efectos de la radiación , Actinas/metabolismo , Animales , Homólogo 4 de la Proteína Discs Large , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neuropéptidos/metabolismo , Ratas , Ratas Wistar , Rayos X/efectos adversosRESUMEN
The direct biological effects of radiation, particularly accelerated heavy particle ions, on neurons are not fully known. Hence, the direct effect of carbon-ion beams on immature neurons was investigated by comparing to the effect of X-rays in vitro using primary hippocampal neurons. Primary neurons were prepared from hippocampi of fetal rats at embryonic day 18 from timed pregnant Wistar rats and cultured with Banker's methods. At 7 Days In Vitro (DIV), the cells were irradiated with 140 kV X-ray and 18.3 MeV/amu carbon-ion beams (LET = 108 keV/µm). The cells were fixed with 4% paraformaldehyde at 12 hours after irradiation. Then, the cells were treated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and DAPI staining for measuring the percentage of apoptosis (apoptotic index: AI). AI in sham-irradiated hippocampal neurons was 18%. The value of AI (AIs) of the cells irradiated with X-rays at 10 or 30 Gy were 15% or 23%, respectively. AI in cells irradiated with carbon-ion beams at 1 Gy, 3 Gy, 5 Gy and 10 Gy were 22%, 23%, 24% and 33%, respectively. AI was significantly increased by carbon-ion beams at 10 Gy (p < 0.001). The apoptosis of hippocampal neurons increased in a dose-dependent manner following both X-ray and carbon-ion beams irradiation. Carbon-ion beams were about 10-fold more effective than X-rays for apoptosis induction in immature hippocampal neurons.
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Apoptosis/efectos de la radiación , Iones Pesados/efectos adversos , Hipocampo/efectos de la radiación , Animales , Carbono , Femenino , Radioterapia de Iones Pesados , Hipocampo/citología , Hipocampo/embriología , Técnicas In Vitro , Neuronas/citología , Neuronas/efectos de la radiación , Embarazo , Tolerancia a Radiación , Ratas , Ratas Wistar , Efectividad Biológica Relativa , Rayos XRESUMEN
Little is known about the direct biological effects of radiation on immature neurons, despite its relevance to the mental retardation caused by irradiation of the brains of fetuses and children. In this study, we investigated the effects of radiation using primary cultured hippocampal neuronal cells with exclusion of glial cells, focusing on cell survival and structural development. Primary neurons were prepared from the hippocampi of fetal rats at embryonic day 18 and cultured according to Banker's methods. After incubation for 7 days, cells were irradiated with X rays and incubated continuously for 7 or 14 days. The number of neurons, their rate of apoptosis, and the patterns of expression of synaptic proteins on the neural dendrites were investigated by immunohistochemical methods. The total numbers of neurons were the same regardless of whether they were irradiated. The number of TUNEL-positive neurons, which can be considered as undergoing apoptosis, increased significantly in a dose-dependent fashion at both 7 and 14 days after irradiation. The mean numbers of clusters of synaptic proteins on neural dendrites, which are considered to represent their developmental level, decreased dose-dependently at both 7 and 14 days after irradiation. These results suggest that radiation not only induces apoptosis but also produces structural defects in the surviving neurons that may directly suppress neural development.
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Hipocampo/efectos de la radiación , Neuronas/citología , Animales , Apoptosis , Técnica del Anticuerpo Fluorescente , Hipocampo/citología , Humanos , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , RatasRESUMEN
Drebrin is a well-known side-binding protein of F-actin in the brain. Immunohistochemical data suggest that the peripheral parts of growing axons are enriched in the drebrin E isoform and mature axons are not. It has also been observed that drebrin E is concentrated in the growth cones of PC12 cells. These data strongly suggest that drebrin E plays a role in axonal growth during development. In this study, we used primary hippocampal neuronal cultures to analyze the role of drebrin E. Immunocytochemistry showed that within axonal growth cones drebrin E specifically localized to the transitional zone, an area in which dense networks of F-actins and microtubules overlapped. Over-expression of drebrin E caused drebrin E and F-actin to accumulate throughout the growth cone and facilitated axonal growth. In contrast, knockdown of drebrin E reduced drebrin E and F-actin in the growth cone and prevented axonal growth. Furthermore, inhibition of myosin II ATPase masked the promoting effects of drebrin E over-expression on axonal growth. These results suggest that drebrin E plays a role in axonal growth through actin-myosin interactions in the transitional zone of axonal growth cones.
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Actinas/metabolismo , Axones/fisiología , Miosinas/metabolismo , Neuropéptidos/fisiología , Actinas/fisiología , Animales , Axones/química , Células Cultivadas , Conos de Crecimiento/química , Conos de Crecimiento/fisiología , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/fisiología , Miosinas/fisiología , Neurogénesis/fisiología , Unión Proteica/fisiología , RatasRESUMEN
Drebrin is a major F-actin-binding protein in the brain. We have recently demonstrated that drebrin A (neurone-specific isoform) clusters at synapses and governs targeting of the post-synaptic density 95 protein to synapses during development. To determine the role of drebrin A on excitatory synapse formation, we analysed whether the suppression of drebrin A expression affects filopodia-spine morphology and synaptic targeting of NMDA receptors in cultured hippocampal neurones. Suppression of developmentally programmed up-regulation of drebrin A by antisense treatment significantly decreased the density and width of filopodia-spines. Immunocytochemistry showed that the antisense treatment did not attenuate synaptic clustering of NMDA receptors under conditions that permitted spontaneous activities but inhibited the accelerated targeting of NMDA receptors into synapses by its antagonist D-(-)-2-amino-5-phosphonopentanoic acid. These results indicate that drebrin A up-regulation plays a pivotal role in spine morphogenesis and activity-dependent synaptic targeting of NMDA receptors.
Asunto(s)
Regulación de la Expresión Génica , Hipocampo/ultraestructura , Neuronas/ultraestructura , Neuropéptidos/genética , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/fisiología , Células Cultivadas , Dendritas/química , Dendritas/ultraestructura , Neuronas/fisiología , Neuropéptidos/análisis , Neuropéptidos/fisiología , Oligonucleótidos Antisentido/farmacología , Receptores de N-Metil-D-Aspartato/análisis , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Sinapsis/química , Sinapsinas/análisisRESUMEN
X-irradiation to neuronal progenitor cells causes brain dysfunctions, such as a mental retardation, in adulthood. However, little has been known about the degree of radiosensitivity of neurons in the developmental stages at which they are most vulnerable. In this study we compared the effect of irradiation on mature neurons with that on immature neurons. Primary dissociated neuronal cultures were prepared from fetal rat hippocampi of embryonic day 18. X-irradiations were performed on the cultured cells at 7 or 21 days in vitro (DIV), and the cells were fixed at 12 or 24 h after irradiation. Then the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The apoptotic changes were measured quantitatively by nuclear pyknosis and DNA fragmentation-both characteristic morphological changes of apoptosis. Light microscopy with differential interference contrast showed that 30 Gy of irradiation increased cellular shrinkage in 7-DIV neurons but not in 21-DIV neurons. Quantitative analysis using DAPI imaging showed that 30 Gy of irradiation significantly enhanced pyknotic changes in 7-DIV neurons after 24 h. In contrast, this irradiation did not enhance any pyknotic changes in 21-DIV neurons after 24 h. Further TUNEL staining also showed that the irradiation did not enhance any DNA fragmentation in nuclei of 21-DIV neurons after 24h. Hence, we showed that the radiosensitivity of 21-DIV postmitotic neurons was significantly lower than that of 7-DIV neurons, indicating that the susceptibility of such neurons depends on their developmental stage.