RESUMEN
The Chinese hamster ovary cell line CHO is widely used for biopharmaceutical production. Genome editing makes it possible to improve the growth properties of cells, their auxotrophy, and the functioning of the apoptosis and autophagy induction systems. Simultaneous editing of multiple genes makes it possible to obtain a cell line with the required genotype faster than several consecutive rounds of genomic knockout, but the probability of success is lower. Simultaneous editing of the dhfr, glul, bak1, and bax genes in the CHO S cells genome yielded 24 clones with signs of auxotrophy for thymidine and glutamine. Five of them turned out to be dhfr+/-, all five contained a knockout of one or two glul alleles. In one clone, 7 out of 8 target alleles were inactivated by a frameshift, and the second dhfr allele was partially inactivated by insertion of the GAA triplet, which reduced the enzyme activity 2.5 times. The probability of simultaneous knockout of both dhfr alleles increased to 50% when the genome was edited with a pair of guide RNAs directed to one exon of the dhfr gene.
Asunto(s)
Edición Génica , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Tetrahidrofolato Deshidrogenasa , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2 , Animales , Células CHO , Cricetinae , Cricetulus , Técnicas de Inactivación de Genes , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Tetrahidrofolato Deshidrogenasa/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genéticaAsunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Investigación Biomédica , Clonación Molecular , Docentes , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Historia del Siglo XX , Historia del Siglo XXIRESUMEN
Obesity is associated with inflammation and insulin resistance (IR), but the regulation of insulin sensitivity (IS) and connections between IS and inflammation remain unclear. We investigated the role of miR-467a-5p, a miRNA induced by hyperglycaemia, in regulating inflammation and blood glucose handling. We previously demonstrated that miR-467a-5p is induced by hyperglycaemia and inhibits the production of thrombospondin-1 (TSP-1), a protein implicated in regulating inflammation. To investigate the role of miR-467 in blood glucose handling and tissue inflammation, WT C57BL/6 mice were fed chow or Western diet from 5 to 32 weeks of age and injected weekly with miR-467a-5p antagonist. Inhibiting miR-467a-5p resulted in 47% increase in macrophage infiltration and increased Il6 levels in adipose tissue, higher plasma insulin levels (98 ng/mL vs 63 ng/mL), and 17% decrease in glucose clearance without increase in weight or HDL/LDL. The antagonist effect was lost in mice on Western diet. Mice lacking TSP-1 lost some but not all of the miR-467 effects, suggesting Thbs1 (and other unknown transcripts) are targeted by miR-467 to regulate inflammation. miR-467a-5p provides a physiological feedback when blood glucose is elevated to avoid inflammation and increased blood glucose and insulin levels, which may prevent IR.
Asunto(s)
Glucemia , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/metabolismo , Insulinas/sangre , MicroARNs/genética , Tejido Adiposo/metabolismo , Animales , Biomarcadores , Citocinas/metabolismo , Modelos Animales de Enfermedad , Glucosa/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Mediadores de Inflamación/metabolismo , Resistencia a la Insulina/genética , Lípidos/sangre , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Especificidad de Órganos , Páncreas/metabolismo , Células RAW 264.7RESUMEN
BACKGROUND: The carotid body (CB) plays a critical role in cyclic intermittent hypoxia (CIH)-induced chemosensitivity; however, the underlying mechanism remains uncertain. We have demonstrated the presence of multiple inotropic glutamate receptors (iGluRs) in CB, and that CIH exposure alters the level of some iGluRs in CB. This result implicates glutamatergic signaling in the CB response to hypoxia. The glutamatergic neurotransmission is not only dependent on glutamate and glutamate receptors, but is also dependent on glutamate transporters, including vesicular glutamate transporters (VGluTs) and excitatory amino acid transporters (EAATs). Here, we have further assessed the expression and distribution of VGluTs and EAATs in human and rat CB and the effect of CIH exposure on glutamate transporters expression. METHODS: The mRNA of VGluTs and EAATs in the human CB were detected by RT-PCR. The protein expression of VGluTs and EAATs in the human and rat CB were detected by Western blot. The distribution of VGluT3, EAAT2 and EAAT3 were observed by immunohistochemistry staining and immunofluorescence staining. Male Sprague-Dawley (SD) rats were exposed to CIH (FIO2 10-21%, 3 min/3 min for 8 h per day) for 2 weeks. The unpaired Student's t-test was performed. RESULTS: Here, we report on the presence of mRNAs for VGluT1-3 and EAAT1-3 in human CB, which is consistent with our previous results in rat CB. The proteins of VGluT1 and 3, EAAT2 and 3, but not VGluT2 and EAAT1, were detected with diverse levels in human and rat CB. Immunostaining showed that VGluT3, the major type of VGluTs in CB, was co-localized with tyrosine hydroxylase (TH) in type I cells. EAAT2 and EAAT3 were distributed not only in type I cells, but also in glial fibrillary acidic protein (GFAP) positive type II cells. Moreover, we found that exposure of SD rats to CIH enhanced the protein level of EAAT3 as well as TH, but attenuated the levels of VGluT3 and EAAT2 in CB. CONCLUSIONS: Our study suggests that glutamate transporters are expressed in the CB, and that glutamate transporters may contribute to glutamatergic signaling-dependent carotid chemoreflex to CIH.
Asunto(s)
Cuerpo Carotídeo/metabolismo , Células Quimiorreceptoras/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/biosíntesis , Proteínas de Transporte Vesicular de Glutamato/biosíntesis , Sistema de Transporte de Aminoácidos X-AG/análisis , Sistema de Transporte de Aminoácidos X-AG/biosíntesis , Sistema de Transporte de Aminoácidos X-AG/genética , Animales , Cuerpo Carotídeo/química , Células Quimiorreceptoras/química , Expresión Génica , Proteínas de Transporte de Glutamato en la Membrana Plasmática/análisis , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas de Transporte Vesicular de Glutamato/análisis , Proteínas de Transporte Vesicular de Glutamato/genéticaRESUMEN
Excitatory amino acid transporter (EAAT)1 and EAAT2 mediate glutamatergic neurotransmission and prevent excitotoxicity through binding and transportation of glutamate into glia. These EAATs may be regulated by metabotropic glutamate receptor 5 (mGluR5), which is also expressed by glia. Whilst we have data from an Affymetrix™ Human Exon 1.0 ST Array showing higher levels of EAAT1 mRNA (+36%) in Brodmann's are (BA)9 of subjects with schizophrenia, there is evidence that EAAT1 and EAAT2, as well as mGluR5 levels, are altered in the cortex of subjects with the disorder. Hence, we measured mRNA levels of these genes in other cortical regions in subjects with that disorder. EAAT1, EAAT2 and mGluR5 mRNA were measured, in triplicate, using Quantitative PCR in BA10 and BA46 from subjects with schizophrenia (n = 20) and age and sex matched controls (n = 18). Levels of mRNA were normalised to the geometric mean of two reference genes, transcription factor B1, mitochondrial (TFB1M) and S-phase kinase-associated protein 1A (SKP1A), for which mRNA did not vary between diagnostic groups in either region. Normalised levels of EAAT1 and EAAT2 mRNA were significantly higher in BA10 (EAAT1: U = 58, p = 0.0002; EAAT2 U = 70, p = 0.0009), but not BA46 (EAAT1: U = 122, p = 0.09; EAAT2: U = 136, p = 0.21), from subjects with schizophrenia compared to controls. mGluR5 levels in BA10 (U = 173, p=0.85) and BA46 (U = 178, p = 0.96) did not vary by cohort. Our data suggests that region-specific increases in cortical EAAT1 and EAAT2 mRNA are involved in schizophrenia pathophysiology and that disrupted glutamate uptake in schizophrenia may be of particular significance in BA10.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores , Esquizofrenia , Sistema de Transporte de Aminoácidos X-AG , Transportador 1 de Aminoácidos Excitadores/genética , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Ácido Glutámico , Humanos , Corteza Prefrontal/metabolismo , ARN Mensajero , Esquizofrenia/genéticaRESUMEN
BACKGROUND: Excitatory amino acid transporter 2 (EAAT2) plays a pivotal role in glutamate clearance in the adult brain, thereby preventing excitotoxic effects. Considering the high efficacy of EAAT2 for glutamate uptake, we hypothesized that the expression of this transporter in mesenchymal stem cells (MSCs) for systemic administration could yield a cell-based glutamate-grabbing therapy, combining the intrinsic properties of these cells with excitotoxic protection. METHODS: To address this hypothesis, EAAT2-encoding cDNA was introduced into MSCs and human embryonic kidney 293 cells (HEK cells) as the control cell line. EAAT2 expression and functionality were evaluated by in vitro assays. Blood glutamate-grabbing activity was tested in healthy and ischemic rat models treated with 3â¯×â¯106 and 9â¯×â¯106 cells/animal. FINDINGS: The expression of EAAT2 in both cell types conferred the expected glutamate-grabbing activity in in vitro and in vivo studies. The functional improvement observed in ischemic rats treated with EAAT2-HEK at low dose, confirmed that this effect was indeed mediated by the glutamate-grabbing activity associated with EAAT2 functionality. Unexpectedly, both cell doses of non-transfected MSCs induced higher protection than transfected EAAT2-MSCs by another mechanism independent of the glutamate-grabbing capacity. INTERPRETATION: Although the transfection procedure most likely interferes with some of the intrinsic protective mechanisms of mesenchymal cells, the results show that the induced expression of EAAT2 in cells represents a novel alternative to mitigate the excitotoxic effects of glutamate and paves the way to combine this strategy with current cell therapies for cerebral ischemia.
Asunto(s)
Isquemia Encefálica/terapia , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Ácido Glutámico/sangre , Células Madre Mesenquimatosas/metabolismo , Animales , Isquemia Encefálica/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Transportador 2 de Aminoácidos Excitadores , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Células HEK293 , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Ratas , TransfecciónRESUMEN
Immune stimulation might be involved in the pathophysiology of major depressive disorder (MDD). This stimulation induces indoleamine 2,3-dioxygenase (IDO), an enzyme that reduces the tryptophan bioavailability to synthesize serotonin. IDO products, kynurenine metabolites, exert neurotoxic/neuroprotective actions through glutamate receptors. Thus, we study elements of these pathways linked to kynurenine metabolite activity examining whether antidepressants (ADs) can modulate them. Male Wistar rats were exposed to chronic mild stress (CMS), and some of them were treated with ADs. The expression of elements of the IDO pathway, including kynurenine metabolites, and their possible modulation by ADs was studied in the frontal cortex (FC). CMS increased IDO expression in FC compared to control group, and ADs restored the IDO expression levels to control values. CMS-induced IDO expression led to increased levels of the excitotoxic quinolinic acid (QUINA) compared to control, and ADs prevented the rise in such levels. Neither CMS nor ADs changed significantly the antiexcitotoxic kynurenic acid (KYNA) levels. The QUINA/KYNA ratio, calculated as excitotoxicity risk indicator, increased after CMS and ADs prevented this increase. CMS lowered excitatory amino acid transporter (EAAT)-1 and EAAT-4 expression, and some ADs restored their expression levels. Furthermore, CMS decreased N-methyl-D-aspartate receptor (NMDAR)-2A and 2B protein expression, and ADs mitigated this decrease. Our research examines the link between CMS-induced pro-inflammatory cytokines and the kynurenine pathway; it shows that CMS alters the kynurenine pathway in rat FC. Importantly, it also reveals the ability of classic ADs to prevent potentially harmful situations related to the brain scenario caused by CMS.
Asunto(s)
Lóbulo Frontal/patología , Lóbulo Frontal/fisiopatología , Ácido Glutámico/metabolismo , Quinurenina/metabolismo , Estrés Psicológico/metabolismo , Estrés Psicológico/fisiopatología , Transmisión Sináptica , Animales , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Enfermedad Crónica , Citocinas/metabolismo , Lóbulo Frontal/efectos de los fármacos , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Mediadores de Inflamación/metabolismo , Ácido Quinurénico/metabolismo , Masculino , Redes y Vías Metabólicas/efectos de los fármacos , Ácido Quinolínico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Estrés Psicológico/tratamiento farmacológico , Transmisión Sináptica/efectos de los fármacos , Triptófano/metabolismoRESUMEN
Evidence demonstrated that the glutamatergic system is implicated in mediating relapse to several drugs of abuse, including methamphetamine (METH). Glutamate homeostasis is maintained by a number of glutamate transporters, such as glutamate transporter type 1 (GLT-1), cystine/glutamate transporter (xCT), and glutamate aspartate transporter (GLAST). In addition, group II metabotropic glutamate receptors (mGluR2/3) were found to be implicated in relapse-seeking behavior. Ample evidence showed that ß-lactam antibiotics are effective in upregulating GLT-1 and xCT expression, thus improving glutamate homeostasis and attenuating relapse to drugs of abuse. In this study, we investigated the reinstatement of METH using conditioned place preference (CPP) in male alcohol-preferring (P) rats exposed to home-cage free choice ethanol drinking. Here, we tested the effect of clavulanic acid (CA), a ß-lactam, on the reinstatement of METH-seeking and ethanol drinking. In addition, we examined the expression of GLT-1, xCT, and GLAST as well as metabotropic glutamate receptor (mGluR2/3) in the nucleus accumbens (NAc) shell, NAc core, and dorsomedial prefrontal cortex (dmPFC). A priming i.p. injection of METH reinstated preference in METH-paired chamber following extinction. Chronic exposure to ethanol decreased the expression of GLT-1 and xCT in the NAc shell, but not in the NAc core or dmPFC. CA treatment blocked the reinstatement of METH-seeking, decreased ethanol intake, and restored the expression of GLT-1 and xCT in the NAc shell. In addition, the expression of mGluR2/3 was increased by CA treatment in the NAc shell and dmPFC. These findings suggest that these glutamate transporters and mGluR2/3 might be potential therapeutic targets for the attenuation of reinstatement to METH-seeking.
Asunto(s)
Trastornos Inducidos por Alcohol/tratamiento farmacológico , Trastornos Relacionados con Anfetaminas/tratamiento farmacológico , Ácido Clavulánico/farmacología , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Trastornos Inducidos por Alcohol/metabolismo , Trastornos Inducidos por Alcohol/fisiopatología , Trastornos Relacionados con Anfetaminas/metabolismo , Trastornos Relacionados con Anfetaminas/fisiopatología , Animales , Ácido Clavulánico/uso terapéutico , Comportamiento de Búsqueda de Drogas , Etanol/toxicidad , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Masculino , Metanfetamina/toxicidad , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Ratas , Receptores de Glutamato Metabotrópico/genética , Recurrencia , Memoria ImplícitaRESUMEN
Cognitive deficits represent core features of schizophrenia, affecting quality of life and functioning. The excitatory amino acid transporter 2 (EAAT2) is responsible for the majority of glutamate reuptake and its activity is crucial for glutamatergic neurotransmission, prevention of excitotoxic damage and cerebral metabolism. Different studies reported that EAAT2 rs4354668 (-181â¯T/G) influences cognitive functions and brain structures in patients with schizophrenia. Specifically, the G allele, linked to lower EAAT2 expression, was associated with impaired prefrontal cognitive performance and reduced grey matter volumes. Cognitive remediation therapy (CRT) is one of the best available tool to treat cognitive deficits in schizophrenia, able to induce a neuroplastic modulation of cognitive functions. The present study aims to investigate the effects of rs4354668 on CRT outcome, also considering possible genotype interaction with antipsychotic (AP) treatment, since EAAT2 expression is negatively influenced by clozapine. We examined rs4354668 in 88 clinically stabilized patients with schizophrenia, treated with CRT and assessed at enrolment, at the end of CRT and after 3â¯months. We observed greater working memory improvements among patients carrying the T/T genotype, regardless of AP treatment. Moreover, we reported a significant interaction between pharmacological treatment and rs4354668 on executive functions, with greater improvements among T/T patients treated with APs other than clozapine. These observations suggest that impaired EAAT2 expression may attenuate CRT outcome. Moreover, our results indicate the possibility that rs4354668 could also differentially influence the response to CRT depending on the AP treatment.
Asunto(s)
Antipsicóticos/farmacología , Disfunción Cognitiva , Remediación Cognitiva/métodos , Función Ejecutiva , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Evaluación de Resultado en la Atención de Salud , Esquizofrenia , Adulto , Disfunción Cognitiva/etiología , Disfunción Cognitiva/genética , Disfunción Cognitiva/terapia , Transportador 2 de Aminoácidos Excitadores , Función Ejecutiva/efectos de los fármacos , Función Ejecutiva/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Estudios Retrospectivos , Esquizofrenia/complicaciones , Esquizofrenia/genética , Esquizofrenia/terapia , Adulto JovenRESUMEN
Targeted expression of genes coding for proteins specific to astrocytes, oligodendrocytes and myelin was performed in frontal cortex area 8 of Creutzfeldt-Jakob disease methionine/methionine and valine/valine (CJD MM1 and VV2, respectively) compared with controls. GFAP (glial fibrillary acidic protein) mRNA was up-regulated whereas SLC1A2 (solute carrier family 1 member 2, coding for glutamate transporter 1: GLT1), AQ4 (aquaporin 4), MPC1 (mitochondrial pyruvate carrier 1) and UCP5 (mitochondrial uncoupled protein 5) mRNAs were significantly down-regulated in CJD MM1 and CJD VV2, and GJA1 (connexin 43) in CJD VV2. OLIG1 and OLIG2 (oligodendocyte transcription factor 1 and 2, respectively), SOX10 (SRY-Box10) and oligodendroglial precursor cell (OPC) marker NG2 (neuronal/glial antigen) 2 were preserved, but GALC (coding for galactosylceramidase), SLC2A1 (solute carrier family 2 member 1: glucose transporter member 1: GLUT1) and MCT1 (monocarboxylic acid transporter 1) mRNA expression levels were significantly reduced in CJD MM1 and CJD VV2. Expression levels of most genes linked to myelin were not altered in the cerebral cortex in CJD. Immunohistochemistry to selected proteins disclosed individual variations but GFAP, Olig-2, AQ4 and GLUT1 correlated with mRNA levels, whereas GLT1 was subjected to individual variations. However, MPC1, UCP5 and MCT1 decrease was more closely related to the respective reduced neuronal immunostaining. These observations support the idea that molecular deficits linked to energy metabolism and solute transport in astrocytes and oligodendrocytes, in addition to neurons, are relevant in the pathogenesis of cortical lesions in CJD.
Asunto(s)
Astrocitos/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Lóbulo Frontal/citología , Oligodendroglía/metabolismo , Transcripción Genética/genética , Astrocitos/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Transportador 2 de Aminoácidos Excitadores , Lóbulo Frontal/patología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Humanos , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Desacopladoras Mitocondriales/genética , Proteínas Desacopladoras Mitocondriales/metabolismo , Transportadores de Ácidos Monocarboxílicos , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos/genética , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Oligodendroglía/patología , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Neuronal hyperactivity is one of the earliest events observed in Alzheimer's disease (AD). Moreover, alterations in the expression of glutamate transporters have been reported to exacerbate amyloid pathology and cognitive deficits in transgenic AD mouse models. However, the molecular links between these pathophysiological changes remain largely unknown. Here, we report novel interaction between presenilin 1 (PS1), the catalytic component of the amyloid precursor protein-processing enzyme, γ-secretase, and a major glutamate transporter-1 (GLT-1). Our data demonstrate that the interaction occurs between PS1 and GLT-1 expressed at their endogenous levels in vivo and in vitro, takes place in both neurons and astrocytes, and is independent of the PS1 autoproteolysis and γ-secretase activity. This intriguing discovery may shed light on the molecular crosstalk between the proteins linked to the maintenance of glutamate homeostasis and Aß pathology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Presenilina-1/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Células Cultivadas , Transportador 2 de Aminoácidos Excitadores/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Humanos , Ratones , Neuronas/metabolismo , Neuronas/patología , Presenilina-1/genética , ProteolisisRESUMEN
The Fibronectin Leucine-Rich Transmembrane protein 2 (FLRT2) has been implicated in several hormone -and sex-dependent physiological and pathological processes (including chondrogenesis, menarche and breast cancer); is known to regulate developmental synapses formation, and is expressed in the hippocampus, a brain structure central for learning and memory. However, the role of FLRT2 in the adult hippocampus and its relevance in sex-dependent brain functions remains unknown. We here used adult single-allele FLRT2 knockout (FLRT2+/-) mice and behavioral, electrophysiological, and molecular/biological assays to examine the effects of FLRT2 haplodeficiency on synaptic plasticity and hippocampus-dependent learning and memory. Female and male FLRT2+/- mice presented morphological features (including body masses, brain shapes/weights, and brain macroscopic cytoarchitectonic organization), indistinguishable from their wild type counterparts. However, in vivo examinations unveiled enhanced hippocampus-dependent spatial memory recall in female FLRT2+/- animals, concomitant with augmented hippocampal synaptic plasticity and decreased levels of the glutamate transporter EAAT2 and beta estrogen receptors. In contrast, male FLRT2+/- animals exhibited deficient memory recall and decreased alpha estrogen receptor levels. These observations propose that FLRT2 can regulate memory functions in the adulthood in a sex-specific manner and might thus contribute to further research on the mechanisms linking sexual dimorphism and cognition.
Asunto(s)
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Plasticidad Neuronal/fisiología , Memoria Espacial/fisiología , Animales , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Transportador 2 de Aminoácidos Excitadores , Femenino , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Hipocampo/metabolismo , Masculino , Ratones , Ratones Noqueados , Plasticidad Neuronal/genética , Factores SexualesRESUMEN
Recently, heterozygous de novo mutations in SCL1A2 have been reported to underlie severe early-onset epileptic encephalopathy. In one male presenting with epileptic seizures and visual impairment, we identified a novel homozygous splicing variant in SCL1A2 (c.1421 + 1G > C) by using exome sequencing. Functional studies on cDNA level confirmed a consecutive loss of function. Our findings suggest that not only de novo mutations but also biallelic variants in SLC1A2 can cause epilepsy and that there is an additional autosomal recessive mode of inheritance. These findings also contribute to the understanding of the genetic mechanism of autosomal dominant SLC1A2-related epileptic encephalopathy as they exclude haploinsufficiency as exclusive genetic mechanism.
Asunto(s)
Epilepsia/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Mutación/genética , Preescolar , Transportador 2 de Aminoácidos Excitadores , Salud de la Familia , Humanos , Masculino , FenotipoRESUMEN
Preterm delivery is associated with neurodevelopmental impairment caused by environmental and genetic factors. Dysfunction of the excitatory amino acid transporter 2 (EAAT2) and the resultant impaired glutamate uptake can lead to neurological disorders. In this study, we investigated the role of single nucleotide polymorphisms (SNPs; g.-200C>A and g.-181A>C) in the EAAT2 promoter in susceptibility to brain injury and neurodisability in very preterm infants born at or before 32-week gestation. DNA isolated from newborns' dried blood spots were used for pyrosequencing to detect both SNPs. Association between EAAT2 genotypes and cerebral palsy, cystic periventricular leukomalacia and a low developmental score was then assessed. The two SNPs were concordant in 89.4% of infants resulting in three common genotypes all carrying two C and two A alleles in different combinations. However, in 10.6% of cases, non-concordance was found, generating six additional rare genotypes. The A alleles at both loci appeared to be detrimental and consequently, the risk of developing cerebral palsy increased four- and sixfold for each additional detrimental allele at -200 and -181 bp, respectively. The two SNPs altered the regulation of the EAAT2 promoter activity and glutamate homeostasis. This study highlights the significance of glutamate in the pathogenesis of preterm brain injury and subsequent development of cerebral palsy and neurodevelopmental disabilities. Furthermore, the described EAAT2 SNPs may be an early biomarker of vulnerability to neurodisability and may aid the development of targeted treatment strategies.
Asunto(s)
Parálisis Cerebral/diagnóstico , Parálisis Cerebral/genética , Variación Genética/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Recien Nacido Prematuro/fisiología , Regiones Promotoras Genéticas/genética , Animales , Astrocitos/patología , Astrocitos/fisiología , Células Cultivadas , Preescolar , Transportador 2 de Aminoácidos Excitadores , Femenino , Humanos , Recién Nacido , Masculino , Polimorfismo de Nucleótido Simple/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Recently, a genome-wide association study of a Caucasian population identified variant rs3794087 in intron 4 of the SLC1A2 gene, which may increase the risk of essential tremor (ET). Considering the overlap in the pathological features and clinical manifestations of ET and Parkinson's disease (PD), several studies on the association between rs3794087 and PD were later performed in other populations. However, results about the role of SLC1A2 rs3794087 in PD were inconsistent. We thus performed a case-control study in a Chinese Han population to investigate the role of SLC1A2 rs3794087 in Chinese patients with PD. Overall, 1096 subjects comprising 546 patients with PD and 550 control subjects were genotyped. A meta-analysis of the data obtained from the current sample-set and those available from prior studies was performed. Taking all patients and controls into consideration, rs3794087 was found to have no significant effect on PD susceptibility in analyses using allelic (pâ¯=â¯.486), genotype (pâ¯=â¯.736), additive (pâ¯=â¯.764), dominant (pâ¯=â¯.438), and recessive (pâ¯=â¯.878) genetic models. The results of the meta-analysis were in agreement with our findings (the pooled OR was 0.97 and 95% CIâ¯=â¯0.85, 1.10). Our study suggested that rs3794087 does not lead to an increased risk of PD in the Chinese Han population. The role of single nucleotide polymorphism rs3794087 in the development of PD remains to be further studied.
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Temblor Esencial/genética , Predisposición Genética a la Enfermedad , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Enfermedad de Parkinson/genética , Polimorfismo de Nucleótido Simple/genética , Pueblo Asiatico/genética , Estudios de Casos y Controles , Transportador 2 de Aminoácidos Excitadores , Femenino , Estudio de Asociación del Genoma Completo/métodos , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Massively parallel sequencing has revealed many de novo mutations in the etiology of developmental and epileptic encephalopathies (EEs), highlighting their genetic heterogeneity. Additional candidate genes have been prioritized in silico by their co-expression in the brain. Here, we evaluate rare coding variability in 20 candidates nominated with the use of a reference gene set of 51 established EE-associated genes. Variants within the 20 candidate genes were extracted from exome-sequencing data of 42 subjects with EE and no previous genetic diagnosis. We identified 7 rare non-synonymous variants in 7 of 20 genes and performed Sanger sequence validation in affected probands and parental samples. De novo variants were found only in SLC1A2 (aka EAAT2 or GLT1) (c.244G>A [p.Gly82Arg]) and YWHAG (aka 14-3-3γ) (c.394C>T [p.Arg132Cys]), highlighting the potential cause of EE in 5% (2/42) of subjects. Seven additional subjects with de novo variants in SLC1A2 (n = 1) and YWHAG (n = 6) were subsequently identified through online tools. We identified a highly significant enrichment of de novo variants in YWHAG, establishing their role in early-onset epilepsy, and we provide additional support for the prior assignment of SLC1A2. Hence, in silico modeling of brain co-expression is an efficient method for nominating EE-associated genes to further elucidate the disorder's etiology and genotype-phenotype correlations.
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Proteínas 14-3-3/genética , Predisposición Genética a la Enfermedad , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Espasmos Infantiles/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Niño , Transportador 2 de Aminoácidos Excitadores , Exoma/genética , Femenino , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Fenotipo , Adulto JovenRESUMEN
Termination of Saccharomyces cerevisiae RNA polymerase II (Pol II) transcripts occurs through two alternative pathways. Termination of mRNAs is coupled to cleavage and polyadenylation while noncoding transcripts are terminated through the Nrd1-Nab3-Sen1 (NNS) pathway in a process that is linked to RNA degradation by the nuclear exosome. Some mRNA transcripts are also attenuated through premature termination directed by the NNS complex. In this paper we present the results of nuclear depletion of the NNS component Nab3. As expected, many noncoding RNAs fail to terminate properly. In addition, we observe that nitrogen catabolite-repressed genes are upregulated by Nab3 depletion.
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Nitrógeno/metabolismo , Proteínas Nucleares/metabolismo , ARN Polimerasa II/genética , ARN Mensajero/genética , ARN Nucleolar Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terminación de la Transcripción Genética/fisiología , Represión Catabólica/genética , Codón sin Sentido/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Glutamato-Amoníaco Ligasa/metabolismo , IMP Deshidrogenasa/biosíntesis , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesisRESUMEN
The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs.
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Ventrículos Laterales/citología , Células-Madre Neurales/citología , Neurogénesis , Neuronas/citología , Proteínas de Transporte Vesicular de Glutamato/metabolismo , Animales , Células Cultivadas , Proteína Doblecortina , Regulación del Desarrollo de la Expresión Génica , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Ventrículos Laterales/metabolismo , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Ratas , Proteínas de Transporte Vesicular de Glutamato/genéticaRESUMEN
The use of antipsychotics carries the risk of metabolic side effects, such as weight gain and new onset type-2 diabetes mellitus. The mechanisms of the observed metabolic alterations are not fully understood. We compared the effects of two atypical antipsychotics, one known to favor weight gain (olanzapine), the other not (aripiprazole), on glucose metabolism. Primary human peripheral blood mononuclear cells (PBMC) were isolated and stimulated with olanzapine or aripiprazole for 72 h. Cellular glucose uptake was analyzed in vitro by 18F-FDG uptake. Further measurements comprised mRNA expression of glucose transporter (GLUT) 1 and 3, GLUT1 protein expression, DNA methylation of GLUT1 promoter region, and proteins involved in downstream glucometabolic processes. We observed a 2-fold increase in glucose uptake after stimulation with aripiprazole. In contrast, olanzapine stimulation decreased glucose uptake by 40%, accompanied by downregulation of the cellular energy sensor AMP activated protein kinase (AMPK). GLUT1 protein expression increased, GLUT1 mRNA expression decreased, and GLUT1 promoter was hypermethylated with both antipsychotics. Pyruvat-dehydrogenase (PDH) complex activity decreased with olanzapine only. Our findings suggest that the atypical antipsychotics olanzapine and aripiprazole differentially affect energy metabolism in PBMC. The observed decrease in glucose uptake in olanzapine stimulated PBMC, accompanied by decreased PDH point to a worsening in cellular energy metabolism not compensated by AMKP upregulation. In contrast, aripiprazole stimulation lead to increased glucose uptake, while not affecting PDH complex expression. The observed differences may be involved in the different metabolic profiles observed in aripiprazole and olanzapine treated patients.
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Antipsicóticos/farmacología , Aripiprazol/farmacología , Benzodiazepinas/farmacología , Glucosa/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Antígenos CD/metabolismo , Metilación de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Transportador 2 de Aminoácidos Excitadores , Citometría de Flujo , Fluorodesoxiglucosa F18 , Regulación de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 3/genética , Transportador de Glucosa de Tipo 3/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Humanos , Cetona Oxidorreductasas/metabolismo , Olanzapina , Proteínas Quinasas/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Estadísticas no ParamétricasRESUMEN
Astrocytes play a fundamental role in maintaining the health and function of the central nervous system. Increasing evidence indicates that astrocytes undergo both cellular and molecular changes at an early stage in neurological diseases, including Alzheimer's disease (AD). These changes may reflect a change from a neuroprotective to a neurotoxic phenotype. Given the lack of current disease-modifying therapies for AD, astrocytes have become an interesting and viable target for therapeutic intervention. The astrocyte transport system covers a diverse array of proteins involved in metabolic support, neurotransmission and synaptic architecture. Therefore, specific targeting of individual transporter families has the potential to suppress neurodegeneration, a characteristic hallmark of AD. A small number of the 400 transporter superfamilies are expressed in astrocytes, with evidence highlighting a fraction of these are implicated in AD. Here, we review the current evidence for six astrocytic transporter subfamilies involved in AD, as reported in both animal and human studies. This review confirms that astrocytes are indeed a viable target, highlights the complexities of studying astrocytes and provides future directives to exploit the potential of astrocytes in tackling AD.