RESUMO
Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis. Our data indicate that LGI1 is expressed at the axonal initial segment and regulates action potential firing by setting the density of the axonal Kv1.1 channels that underlie dendrotoxin-sensitive D-type potassium current. LGI1 deficiency incurs a >50% down-regulation of the expression of Kv1.1 and Kv1.2 via a posttranscriptional mechanism, resulting in a reduction in the capacity of axonal D-type current to limit glutamate release, thus contributing to epileptogenesis.
Assuntos
Axônios/metabolismo , Proteínas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Potenciais de Ação , Animais , Venenos Elapídicos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Canal de Potássio Kv1.2/metabolismo , Camundongos Mutantes , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Proteínas/genética , Proteínas/farmacologia , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
Cumulative evidence indicates that MYC, one of the major downstream effectors of NOTCH1, is a critical component of T-cell acute lymphoblastic leukemia (T-ALL) oncogenesis and a potential candidate for targeted therapy. However, MYC is a complex oncogene, involving both fine protein dosage and cell-context dependency, and detailed understanding of MYC-mediated oncogenesis in T-ALL is still lacking. To better understand how MYC is interspersed in the complex T-ALL oncogenic networks, we performed a thorough molecular and biochemical analysis of MYC activation in a comprehensive collection of primary adult and pediatric patient samples. We find that MYC expression is highly variable, and that high MYC expression levels can be generated in a large number of cases in absence of NOTCH1/FBXW7 mutations, suggesting the occurrence of multiple activation pathways in addition to NOTCH1. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down-regulation of PTEN, and that NOTCH1(m) might play a dual transcriptional and posttranscriptional role in this process. Altogether, our data lend further support to the significance of therapeutic targeting of MYC and/or the PTEN/AKT pathways, both in GSI-resistant and identified NOTCH1-independent/MYC-mediated T-ALL patients.
Assuntos
Genes myc , PTEN Fosfo-Hidrolase/fisiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adulto , Células Cultivadas , Criança , Regulação Leucêmica da Expressão Gênica , Humanos , Células Jurkat , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Processamento Pós-Transcricional do RNA/genética , Processamento Pós-Transcricional do RNA/fisiologia , Transdução de Sinais/genética , Ativação Transcricional/genética , TransfecçãoRESUMO
Molecular minimal residual disease (MRD) analysis is fast emerging as an essential clinical decision-making tool for the treatment and follow-up of mature B cell malignancies. Current EuroMRD consensus IGH real-time quantitative polymerase chain reaction RQ-PCR assays rely on flow cytometric assessment of diagnostic tumour burdens to construct 'normalized', patient-specific, diagnostic DNA-based MRD quantification standards. Here, we propose a new 'hybrid' assay that relies on plasmid-based quantification of patient-specific IGH VDJ targets by consensus IGH real time (RQ)-PCR, combined with EuroMRD guidelines, for MRD monitoring in lymphoid malignancies. This assay was evaluated for MRD assessment in a total of 273 samples from 29 mantle cell lymphoma (MCL) patients treated within a Groupe Ouest Est d'Etude des Leucémies et Autres Maladies du Sang (GOELAMS) Phase II trial and was feasible, reliable and consistently comparable to gold-standard MRD techniques (99% concordance across all samples including 32 samples within the quantitative range) when analysed in parallel (117 samples). Integrating clinical prognostic parameters and MRD status in peripheral blood at the post-induction stage was predictive of progression-free survival (P = 0·034) thus demonstrating the clinical utility of the approach. Plasmid-based standards for the quantification of IGH VDJ targets are therefore confirmed to offer new opportunities for further standardization and clinical evaluation of MRD-guided management of patients with mature B cell malignancies.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Linfoma de Célula do Manto/diagnóstico , Recombinação V(D)J/genética , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Intervalo Livre de Doença , Estudos de Viabilidade , Feminino , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Humanos , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Plasmídeos/genética , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
BACKGROUND: Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1alpha and HIF-1beta/aryl hydrocarbon receptor nuclear translocator (ARNT) subunits. The prognostic relevance of HIF-1alpha protein overexpression has been shown in breast cancer. The impact of HIF-1alpha alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known. METHODS: Using real-time quantitative reverse transcription PCR assays, we measured mRNA concentrations of total HIF-1alpha and 4 variants in breast tissue specimens in a series of 29 normal tissues or benign lesions (normal/benign) and 53 primary carcinomas. In breast cancers HIF-1alpha splice variant levels were compared to clinicopathological parameters including tumour microvessel density and metastasis-free survival. RESULTS: HIF-1alpha isoforms containing a three base pairs TAG insertion between exon 1 and exon 2 (designated HIF-1alphaTAG) and HIF-1alpha736 mRNAs were found expressed at higher levels in oestrogen receptor (OR)-negative carcinomas compared to normal/benign tissues (P = 0.009 and P = 0.004 respectively). In breast carcinoma specimens, lymph node status was significantly associated with HIF-1alphaTAG mRNA levels (P = 0.037). Significant statistical association was found between tumour grade and HIF-1alphaTAG (P = 0.048), and total HIF-1alpha (P = 0.048) mRNA levels. HIF-1alphaTAG mRNA levels were also inversely correlated with both oestrogen and progesterone receptor status (P = 0.005 and P = 0.033 respectively). Univariate analysis showed that high HIF-1alphaTAG mRNA levels correlated with shortened metastasis free survival (P = 0.01). CONCLUSIONS: Our results show for the first time that mRNA expression of a HIF-1alphaTAG splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis.See commentary: http://www.biomedcentral.com/1741-7015/8/45.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/secundário , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metástase Neoplásica/diagnóstico , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Adolescente , Adulto , Idoso , Biomarcadores , Criança , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Most neuronal types have a well-identified electrical phenotype. It is now admitted that a same phenotype can be produced using multiple biophysical solutions defined by ion channel expression levels. This argues that systems-level approaches are necessary to understand electrical phenotype genesis and stability. Midbrain dopaminergic (DA) neurons, although quite heterogeneous, exhibit a characteristic electrical phenotype. However, the quantitative genetic principles underlying this conserved phenotype remain unknown. Here we investigated the quantitative relationships between ion channels' gene expression levels in midbrain DA neurons using single-cell microfluidic qPCR. Using multivariate mutual information analysis to decipher high-dimensional statistical dependences, we unravel co-varying gene modules that link neurotransmitter identity and electrical phenotype. We also identify new segregating gene modules underlying the diversity of this neuronal population. We propose that the newly identified genetic coupling between neurotransmitter identity and ion channels may play a homeostatic role in maintaining the electrophysiological phenotype of midbrain DA neurons.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica/genética , Canais Iônicos/genética , Neurotransmissores/genética , Animais , Dopamina/genética , Dopamina/metabolismo , Fenômenos Eletrofisiológicos , Canais Iônicos/metabolismo , Mesencéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Neurotransmissores/metabolismo , Substância Negra/metabolismo , Área Tegmentar Ventral/metabolismoRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs of approximately 18 to 25 nucleotides in length that negatively regulate gene expression via either the degradation or translational inhibition of their target mRNAs. Because miRNAs are essential for the regulation of critical physiological processes as well as a variety of pathological events, they have emerged as a novel class of molecular diagnostic biomarkers and therapeutic agents or targets. Accordingly, the need for novel methods for the quantification of miRNA has increased due to interest in their clinical implications. Currently, real-time quantitative polymerase chain reaction (qPCR) is considered the most robust technology for nucleic acid quantification. Different tools for miRNA quantification by using qPCR are now commercially available, but only relative quantification strategies have been reported. This situation may be partly due to the difficulty in obtaining an appropriate molecule with which to establish an miRNA calibration range. Here, we describe a rapid and convenient strategy for the development of a calibrator, which enables the absolute quantification of miRNAs by using qPCR and allows the cloning of a synthetic sequence of interest instead of a PCR product into a plasmid.
Assuntos
MicroRNAs/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Humanos , Técnicas In VitroRESUMO
Determination of T-cell clonality has an important additional value for diagnosis of T-cell lymphomas. Various molecular methods have been developed, including polymerase chain reaction (PCR) of T-cell receptor γ (TCRγ). The detection of PCR products usually relies commonly on either GeneScan (GS) analysis or heteroduplex (HD) analysis by polyacrylamide gel electrophoresis (PAGE). These techniques have their disadvantages, being relatively time-consuming and laborious or requiring expensive equipment. Here, we propose an alternative method that combines multiplex PCR and HD analysis by microcapillary electrophoresis (ME) on the Agilent 2100 Bioanalyzer. The sensitivity of the method was determined with clonal PEER T-cell line DNA dilution in polyclonal DNA and was evaluated as 1-5%. Fifty-three samples from patients with T-cell lymphoproliferative disorders were analyzed by HD analysis using ME and GS analyses. Comparison of the two techniques showed them to be highly concordant (93% similarity). The rate of clonality detection by GS analysis was higher than HD analysis by ME, but none of the discordant patients (n=5) has yet developed lymphoma. HD analysis by ME to reveal TCRγ gene rearrangements in clinical specimens was consistent with clinical data and the outcome of patients. Detection of T-cell clonality by HD analysis with ME is sensitive, practical, safe and represents a potential alternative to PAGE and GS analysis.
Assuntos
Rearranjo Gênico da Cadeia gama dos Receptores de Antígenos dos Linfócitos T , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T/genética , Análise Heteroduplex/métodos , Células Clonais/metabolismo , Eficiência , Eletroforese Capilar/métodos , Humanos , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Microquímica/métodos , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
The aim of the study is to compare the expression level of candidate genes between patients suffering from a severe major depressive episode (MDE) and controls, and also among patients during MDE evolution. After a comprehensive review of the biological data related to mood disorders, we initiated a hypothesis-driven exploration of candidate mRNAs. Using RT-qPCR, we analyzed peripheral blood mononuclear cells (PBMCs) mRNA obtained from a homogeneous population of 11 patients who suffered from severe melancholic MDE. To assess the evolution of MDE, we analyzed PBMC mRNAs that were collected on Day 1 and 8 weeks later. Data from these patient samples were analyzed in comparison to age- and sex-matched healthy controls. Among 40 candidate genes consistently transcribed in PBMCs, 10 were differentially expressed in at least one comparison. We found that variations of mRNA levels for NRG1, SORT1 and TPH1 were interesting state-dependent biological markers of the disease. We also observed that variations in other mRNA expression were associated with treatment efficacy or clinical improvement (CREB1, HDAC5, HSPA2, HTR1B, HTR2A, and SLC6A4/5HTT). Significantly, 5HTT exhibited a strong correlation with clinical score evolution. We also found a state-independent marker, IL10. Moreover, the analysis of 2 separate MDEs concerning a same patient revealed comparable results for the expression of CREB1, HSPA2, HTR1B, NRG1 and TPH1. Overall, our results indicate that PBMCs obtained at different time points during MDE progression represent a promising avenue to discover biological markers for depression.
Assuntos
Biomarcadores/sangue , Transtorno Depressivo Maior , Regulação da Expressão Gênica/genética , Leucócitos Mononucleares/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Idoso , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Estudos de Casos e Controles , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/genética , Transtorno Depressivo Maior/fisiopatologia , Progressão da Doença , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Masculino , Pessoa de Meia-Idade , Neuregulina-1/genética , Neuregulina-1/metabolismo , Escalas de Graduação Psiquiátrica , RNA Mensageiro/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Estatísticas não Paramétricas , Fatores de Tempo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismoRESUMO
BACKGROUND: Familial dysautonomia (FD) is a hereditary neuropathy caused by mutations in the IKBKAP gene, the most common of which results in variable tissue-specific mRNA splicing with skipping of exon 20. Defective splicing is especially severe in nervous tissue, leading to incomplete development and progressive degeneration of sensory and autonomic neurons. The specificity of neuron loss in FD is poorly understood due to the lack of an appropriate model system. To better understand and modelize the molecular mechanisms of IKBKAP mRNA splicing, we collected human olfactory ecto-mesenchymal stem cells (hOE-MSC) from FD patients. hOE-MSCs have a pluripotent ability to differentiate into various cell lineages, including neurons and glial cells. METHODOLOGY/PRINCIPAL FINDINGS: We confirmed IKBKAP mRNA alternative splicing in FD hOE-MSCs and identified 2 novel spliced isoforms also present in control cells. We observed a significant lower expression of both IKBKAP transcript and IKAP/hELP1 protein in FD cells resulting from the degradation of the transcript isoform skipping exon 20. We localized IKAP/hELP1 in different cell compartments, including the nucleus, which supports multiple roles for that protein. We also investigated cellular pathways altered in FD, at the genome-wide level, and confirmed that cell migration and cytoskeleton reorganization were among the processes altered in FD. Indeed, FD hOE-MSCs exhibit impaired migration compared to control cells. Moreover, we showed that kinetin improved exon 20 inclusion and restores a normal level of IKAP/hELP1 in FD hOE-MSCs. Furthermore, we were able to modify the IKBKAP splicing ratio in FD hOE-MSCs, increasing or reducing the WT (exon 20 inclusion):MU (exon 20 skipping) ratio respectively, either by producing free-floating spheres, or by inducing cells into neural differentiation. CONCLUSIONS/SIGNIFICANCE: hOE-MSCs isolated from FD patients represent a new approach for modeling FD to better understand genetic expression and possible therapeutic approaches. This model could also be applied to other neurological genetic diseases.