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1.
Endocrinology ; 159(4): 1844-1859, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29509880

RESUMO

Pituitary growth hormone (GH) and insulinlike growth factor (IGF)-1 are anabolic hormones whose physiological roles are particularly important during development. The activity of the GH/IGF-1 axis is controlled by complex neuroendocrine systems including two hypothalamic neuropeptides, GH-releasing hormone (GHRH) and somatostatin (SRIF), and a gastrointestinal hormone, ghrelin. The neurotransmitter acetylcholine (ACh) is involved in tuning GH secretion, and its GH-stimulatory action has mainly been shown in adults but is not clearly documented during development. ACh, together with these hormones and their receptors, is expressed before birth, and somatotroph cells are already responsive to GHRH, SRIF, and ghrelin. We thus hypothesized that ACh could contribute to the modulation of the main components of the somatotropic axis during development. In this study, we generated a choline acetyltransferase knockout mouse line and showed that heterozygous mice display a transient deficit in ACh from embryonic day 18.5 to postnatal day 10, and they recover normal ACh levels from the second postnatal week. This developmental ACh deficiency had no major impact on weight gain and cardiorespiratory status of newborn mice. Using this mouse model, we found that endogenous ACh levels determined the concentrations of circulating GH and IGF-1 at embryonic and postnatal stages. In particular, serum GH level was correlated with brain ACh content. ACh also modulated the levels of GHRH and SRIF in the hypothalamus and ghrelin in the stomach, and it affected the levels of these hormones in the circulation. This study identifies ACh as a potential regulator of the somatotropic axis during the developmental period.


Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/metabolismo , Hormônio do Crescimento/sangue , Hipotálamo/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Hipófise/metabolismo , Acetilcolina/sangue , Animais , Colina O-Acetiltransferase/genética , Mucosa Gástrica/metabolismo , Grelina/metabolismo , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Heterozigoto , Camundongos , Camundongos Knockout , Sistemas Neurossecretores/metabolismo
3.
Mol Metab ; 6(1): 159-172, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28123946

RESUMO

The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity. OBJECTIVE: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly. METHODS: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors. RESULTS: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation. CONCLUSIONS: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.


Assuntos
Proteínas de Transporte/metabolismo , Obesidade/metabolismo , Receptores para Leptina/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Peso Corporal/fisiologia , Proteínas de Transporte/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leptina/metabolismo , Leptina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Receptores para Leptina/fisiologia , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos
4.
Nat Biotechnol ; 34(7): 746-51, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27272383

RESUMO

Widespread application of gene therapy will depend on the development of simple methods to regulate the expression of therapeutic genes. Here we harness an endogenous signaling pathway to regulate therapeutic gene expression through diet. The GCN2-eIF2α signaling pathway is specifically activated by deficiencies in any essential amino acid (EAA); EAA deficiency leads to rapid expression of genes regulated by ATF4-binding cis elements. We found that therapeutic genes under the control of optimized amino acid response elements (AAREs) had low basal expression and high induced expression. We applied our system to regulate the expression of TNFSF10 (TRAIL) in the context of glioma therapy and found that intermittent activation of this gene by EEA-deficient meals retained its therapeutic efficacy while abrogating its toxic effects on normal tissue. The GCN2-eIF2α pathway is expressed in many tissues, including the brain, and is highly specific to EAA deficiency. Our system may be particularly well suited for intermittent regulation of therapeutic transgenes over short or long time periods.


Assuntos
Aminoácidos Essenciais/administração & dosagem , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Administração Oral , Aminoácidos Essenciais/farmacocinética , Animais , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Ingestão de Alimentos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Transgenes/genética , Resultado do Tratamento
5.
J Chem Neuroanat ; 76(Pt A): 48-60, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26744118

RESUMO

The present study was intended to combine three therapeutic approaches in a well-defined rat model of spinal cord injury, a lateral hemisection at thoracic level. A guidance channel was implanted at the lesion site. This channel was seeded with native Schwann cells or Schwann cells that had been previously transduced with a lentiviral vector carrying the GDNF gene. Thereafter, these experiences were reproduced in animals injected with lentiviral vectors carrying a shRNA for GFAP (Lv-shGFAP), which has recently been shown to block glial scar formation. Functional evaluations showed that Lv-shGFAP induced a significant improvement in recovery in animals grafted with Schwann cells. Histological studies demonstrated the outgrowth of axons in the guidance channel containing Schwann cells transduced or not with GDNF. This axonal growth was enhanced in rats receiving Lv-shGFAP vector. Also, a significant increase of serotonergic innervation of the injured hemicord, distal to the lesion, was found only in animals treated with Lv-shGFAP vectors. Importantly, this study confirms that glial scar formation is a major impediment for axonal sprouting after spinal cord injury, and emphasizes the importance of serotonergic innervation for locomotor function. Moreover we show a significant additive effect of a combinatorial approach to axonal regeneration in the injured spinal cord.


Assuntos
Neuroglia/patologia , Células de Schwann/transplante , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Animais , Sobrevivência Celular , Cicatriz/patologia , Feminino , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Lentivirus/genética , Locomoção , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Neurônios Serotoninérgicos/fisiologia , Medula Espinal/patologia
6.
Sci Signal ; 8(374): rs5, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25921292

RESUMO

The eIF2α-ATF4 pathway is involved in cellular adaptation to stress and is dysregulated in numerous diseases. Activation of this pathway leads to phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and the recruitment of the transcription factor ATF4 (activating transcription factor 4) to specific CCAAT/enhancer binding protein (C/EBP)-ATF response elements (CAREs) located in the promoters of target genes. To monitor the spatiotemporal modulation of this pathway in living animals, we generated a novel CARE-driven luciferase mouse model (CARE-LUC). These transgenic mice enable the investigation of the eIF2α-ATF4 pathway activity in the whole organism and at the tissue and cellular levels by combining imaging, luciferase assays, and immunochemistry. Using this mouse line, we showed the tissue-specific activation pattern of this pathway in response to amino acid deficiency or endoplasmic reticulum stress and the hepatic induction of this pathway in a stress-related pathology model of liver fibrosis. The CARE-LUC mouse model represents an innovative tool to investigate the eIF2α-ATF4 axis and to develop drugs targeting this important pathway in the remediation of related pathologies.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Imagem Molecular , Transdução de Sinais , Estresse Fisiológico , Fator 4 Ativador da Transcrição/genética , Animais , Fator de Iniciação 2 em Eucariotos/genética , Camundongos , Camundongos Transgênicos
7.
J Neurosci Res ; 93(1): 43-55, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25131829

RESUMO

In spinal cord injury (SCI), absence of functional recovery and lack of spontaneous axonal regeneration are attributed, among other factors, to the formation of a glial scar that forms both physical and chemical barriers. The glial scar is composed mainly of reactive astrocytes that overexpress two intermediate filament proteins, glial fibrillary acidic protein (GFAP) and vimentin (VIM). To promote regeneration and sprouting of spared axons after spinal cord trauma and with the objective of translation to clinics, we designed an original in vivo gene transfer strategy to reduce glial scar formation after SCI, based on the RNA interference (RNAi)-mediated inhibition of GFAP and VIM. We first show that direct injection of lentiviral vectors expressing short hairpin RNA (shRNA) against GFAP and VIM in a mouse model of SCI allows efficient and specific targeting of astrocytes. We then demonstrate that the lentiviral-mediated and stable expression of shGFAP and shVIM leads to a strong reduction of astrogliosis, improves functional motor recovery, and promotes axonal regrowth and sprouting of spared axons. This study thus examplifies how the nonneuronal environment might be a major target within the lesioned central nervous system to promote axonal regeneration (and sprouting) and validates the use of lentiviral-mediated RNAi in SCI.


Assuntos
Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/terapia , Vimentina/metabolismo , Análise de Variância , Animais , Astrócitos/metabolismo , Axônios/fisiologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos/fisiologia , Proteína Glial Fibrilar Ácida/genética , Lentivirus/genética , Locomoção/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Serotonina/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Vimentina/genética
8.
Dev Neurobiol ; 75(7): 721-37, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25369423

RESUMO

Motor activities are controlled by neural networks in the ventral spinal cord and consist in motor neurons and a set of distinct cardinal classes of spinal interneurons. These interneurons arise from distinct progenitor domains (p0-p3) delineated according to a transcriptional code. Neural progenitors of each domain express a unique combination of transcription factors (TFs) that largely contribute to determine the fate of four classes of interneurons (V0-V3) and motor neurons. In p2 domain, at least four subtypes of interneurons namely V2a, V2b, V2c, and Pax6(+) V2 are generated. Although genetic and molecular mechanisms that specify V2a and V2b are dependent on complex interplay between several TFs including Nkx6.1, Irx3, Gata2, Foxn4, and Ascl1, and signaling pathways such as Notch and TGF-ß, the sequence order of the activation of these regulators and their respective contribution are not completely elucidated yet. Here, we provide evidence by loss- or gain-of-function experiments that Gata2 is necessary for the normal development of both V2a and V2b neurons. We demonstrate that Nkx6.1 and Dll4 positively regulate the activation of Gata2 and Foxn4 in p2 progenitors. Gata2 also participates in the maintenance of p2 domain by repressing motor neuron differentiation and exerting a feedback control on patterning genes. Finally, Gata2 promotes the selective activation of V2b program at the expense of V2a fate. Thus our results provide new insights on the hierarchy and complex interactions between regulators of V2 genetic program.


Assuntos
Proteínas Aviárias/metabolismo , Fator de Transcrição GATA2/metabolismo , Interneurônios/fisiologia , Medula Espinal/embriologia , Medula Espinal/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação ao Cálcio , Embrião de Galinha , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA2/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Interneurônios/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Neurogênese/fisiologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Medula Espinal/citologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo
9.
PLoS One ; 9(8): e104896, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25118945

RESUMO

Epidemiological findings indicate that transient environmental influences during perinatal life, especially nutrition, may have deleterious heritable health effects lasting for the entire life. Indeed, the fetal organism develops specific adaptations that permanently change its physiology/metabolism and that persist even in the absence of the stimulus that initiated them. This process is termed "nutritional programming". We previously demonstrated that mothers fed a Low-Protein-Diet (LPD) during gestation and lactation give birth to F1-LPD animals presenting metabolic consequences that are different from those observed when the nutritional stress is applied during gestation only. Compared to control mice, adult F1-LPD animals have a lower body weight and exhibit a higher food intake suggesting that maternal protein under-nutrition during gestation and lactation affects the energy metabolism of F1-LPD offspring. In this study, we investigated the origin of this apparent energy wasting process in F1-LPD and demonstrated that minimal energy expenditure is increased, due to both an increased mitochondrial function in skeletal muscle and an increased mitochondrial density in White Adipose Tissue. Importantly, F1-LPD mice are protected against high-fat-diet-induced obesity. Clearly, different paradigms of exposure to malnutrition may be associated with differences in energy expenditure, food intake, weight and different susceptibilities to various symptoms associated with metabolic syndrome. Taken together these results demonstrate that intra-uterine environment is a major contributor to the future of individuals and disturbance at a critical period of development may compromise their health. Consequently, understanding the molecular mechanisms may give access to useful knowledge regarding the onset of metabolic diseases.


Assuntos
Metabolismo Energético/genética , Lactação/fisiologia , Mitocôndrias/fisiologia , Obesidade/prevenção & controle , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Deficiência de Proteína/fisiopatologia , Tecido Adiposo Branco/metabolismo , Fatores Etários , Animais , Western Blotting , Temperatura Corporal , Peso Corporal , Calorimetria Indireta , Primers do DNA/genética , Dieta Hiperlipídica/efeitos adversos , Ingestão de Alimentos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Obesidade/metabolismo , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
PLoS One ; 9(6): e99649, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24956106

RESUMO

Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable.


Assuntos
Bacteriófagos/metabolismo , Dano ao DNA , Vetores Genéticos/metabolismo , Hibridização Genética , Lentivirus/genética , Recombinação Genética , Animais , Sítios de Ligação Microbiológicos/genética , Sequência de Bases , Linhagem Celular , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Insercional/genética , Sinais de Localização Nuclear , Reação em Cadeia da Polimerase
11.
Mol Genet Genomic Med ; 2(2): 152-9, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24689078

RESUMO

GPR88, coding for a G protein-coupled orphan receptor that is highly represented in the striatum, is a strong functional candidate gene for neuropsychiatric disorders and is located at 1p22-p21, a chromosomal region that we have previously linked to bipolar disorder (BD) in the Sardinian population. In order to ascertain the relevance of GPR88 as a risk factor for psychiatric diseases, we performed a genetic association analysis between GPR88 and BD in a sample of triads (patient and both parents) recruited in the Sardinian and the Palestinian population as well as between GPR88 and schizophrenia (SZ) in triads from the Xhosa population in South Africa. We found a positive association between GPR88 and BD in the Sardinian and Palestinian triads. Moreover, we found a positive association between GPR88 and SZ in triads from the Xhosa population in South Africa. When these results were corrected for multiple testing, the association between GPR88 and BD was maintained in the Palestinian population. Thus, these results suggest that GPR88 deserves consideration as a candidate gene for psychiatric diseases and requires to be further investigated in other populations.

12.
Neurobiol Dis ; 65: 102-11, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24486622

RESUMO

Motor neuron diseases are characterized by the selective chronic dysfunction of a subset of motor neurons and the subsequent impairment of neuromuscular function. To reproduce in the mouse these hallmarks of diseases affecting motor neurons, we generated a mouse line in which ~40% of motor neurons in the spinal cord and the brainstem become unable to sustain neuromuscular transmission. These mice were obtained by conditional knockout of the gene encoding choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine. The mutant mice are viable and spontaneously display abnormal phenotypes that worsen with age including hunched back, reduced lifespan, weight loss, as well as striking deficits in muscle strength and motor function. This slowly progressive neuromuscular dysfunction is accompanied by muscle fiber histopathological features characteristic of neurogenic diseases. Unexpectedly, most changes appeared with a 6-month delay relative to the onset of reduction in ChAT levels, suggesting that compensatory mechanisms preserve muscular function for several months and then are overwhelmed. Deterioration of mouse phenotype after ChAT gene disruption is a specific aging process reminiscent of human pathological situations, particularly among survivors of paralytic poliomyelitis. These mutant mice may represent an invaluable tool to determine the sequence of events that follow the loss of function of a motor neuron subset as the disease progresses, and to evaluate therapeutic strategies. They also offer the opportunity to explore fundamental issues of motor neuron biology.


Assuntos
Acetilcolina/metabolismo , Colina O-Acetiltransferase/deficiência , Doença dos Neurônios Motores/patologia , Neurônios Motores/metabolismo , Fatores Etários , Análise de Variância , Animais , Peso Corporal/genética , Colina O-Acetiltransferase/genética , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Feminino , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Transgênicos , Doença dos Neurônios Motores/genética , Neurônios Motores/classificação , Força Muscular/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Fatores Sexuais
13.
PLoS One ; 8(8): e71363, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977029

RESUMO

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients.


Assuntos
Animais Geneticamente Modificados , Heterogeneidade Genética , Guanilato Ciclase/genética , Células Fotorreceptoras Retinianas Cones/patologia , Distrofias Retinianas/genética , Transgenes , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Eletrorretinografia , Genes Dominantes , Vetores Genéticos , Guanilato Ciclase/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Lentivirus/genética , Dados de Sequência Molecular , Mutação , Fenótipo , Células Fotorreceptoras Retinianas Cones/enzimologia , Distrofias Retinianas/patologia , Homologia de Sequência de Aminoácidos , Índice de Gravidade de Doença , Suínos/genética , Acuidade Visual
14.
Mol Ther Nucleic Acids ; 2: e75, 2013 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-23403517

RESUMO

The rapamycin-inducible gene regulation system was designed to minimize immune reactions in man and may thus be suited for gene therapy. We assessed whether this system indeed induces no immune responses. The protein components of the regulation system were produced in the human cell lines HEK 293T, D407, and HER 911 following lentiviral transfer of the corresponding genes. Stable cell lines were established, and the peptides presented by major histocompatibility complex class I (MHC I) molecules on transduced and wild-type (wt) cells were compared by differential mass spectrometry. In all cell lines examined, expression of the transgenes resulted in prominent changes in the repertoire of MHC I-presented self-peptides. No MHC I ligands originating from the transgenic proteins were detected. In vitro analysis of immunogenicity revealed that transduced D407 cells displayed slightly higher capacity than wt controls to promote proliferation of cytotoxic T cells. These results indicate that therapeutic manipulations within the genome of target cells may affect pathways involved in the processing of peptide antigens and their presentation by MHC I. This makes the genomic modifications visible to the immune system which may recognize these events and respond. Ultimately, the findings call attention to a possible immune risk.Molecular Therapy - Nucleic Acids (2013) 2, e75; doi:10.1038/mtna.2013.3; published online 12 February 2013.

15.
Glia ; 61(2): 225-39, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23047160

RESUMO

Glioblastoma multiform (GBM) are devastating brain tumors containing a fraction of multipotent stem-like cells which are highly tumorigenic. These cells are resistant to treatments and are likely to be responsible for tumor recurrence. One approach to eliminate GBM stem-like cells would be to force their terminal differentiation. During development, neurons formation is controlled by neurogenic transcription factors such as Ngn1/2 and NeuroD1. We found that in comparison with oligodendrogenic genes, the expression of these neurogenic genes is low or absent in GBM tumors and derived cultures. We thus explored the effect of overexpressing these neurogenic genes in three CD133(+) Sox2(+) GBM stem-like cell cultures and the U87 glioma line. Introduction of Ngn2 in CD133(+) cultures induced massive cell death, proliferation arrest and a drastic reduction of neurosphere formation. Similar effects were observed with NeuroD1. Importantly, Ngn2 effects were accompanied by the downregulation of Olig2, Myc, Shh and upregulation of Dcx and NeuroD1 expression. The few surviving cells adopted a typical neuronal morphology and some of them generated action potentials. These cells appeared to be produced at the expense of GFAP(+) cells which were radically reduced after differentiation with Ngn2. In vivo, Ngn2-expressing cells were unable to form orthotopic tumors. In the U87 glioma line, Ngn2 could not induce neuronal differentiation although proliferation in vitro and tumoral growth in vivo were strongly reduced. By inducing cell death, cell cycle arrest or differentiation, this work supports further exploration of neurogenic proteins to oppose GBM stem-like and non-stem-like cell growth.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/farmacologia , Neoplasias Encefálicas/patologia , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Fatores de Transcrição/farmacologia , Antígeno AC133 , Antígenos CD/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Morte Celular , Citometria de Fluxo , Proteína Glial Fibrilar Ácida/metabolismo , Glicoproteínas/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Fator de Transcrição 2 de Oligodendrócitos , Proteína Oncogênica p55(v-myc)/metabolismo , Peptídeos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transfecção , Células Tumorais Cultivadas
16.
Am J Psychiatry ; 169(9): 963-73, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22885689

RESUMO

OBJECTIVE: The authors used a genome-wide association study (GWAS) of multiply affected families to investigate the association of schizophrenia to common single-nucleotide polymorphisms (SNPs) and rare copy number variants (CNVs). METHOD: The family sample included 2,461 individuals from 631 pedigrees (581 in the primary European-ancestry analyses). Association was tested for single SNPs and genetic pathways. Polygenic scores based on family study results were used to predict case-control status in the Schizophrenia Psychiatric GWAS Consortium (PGC) data set, and consistency of direction of effect with the family study was determined for top SNPs in the PGC GWAS analysis. Within-family segregation was examined for schizophrenia-associated rare CNVs. RESULTS: No genome-wide significant associations were observed for single SNPs or for pathways. PGC case and control subjects had significantly different genome-wide polygenic scores (computed by weighting their genotypes by log-odds ratios from the family study) (best p=10(-17), explaining 0.4% of the variance). Family study and PGC analyses had consistent directions for 37 of the 58 independent best PGC SNPs (p=0.024). The overall frequency of CNVs in regions with reported associations with schizophrenia (chromosomes 1q21.1, 15q13.3, 16p11.2, and 22q11.2 and the neurexin-1 gene [NRXN1]) was similar to previous case-control studies. NRXN1 deletions and 16p11.2 duplications (both of which were transmitted from parents) and 22q11.2 deletions (de novo in four cases) did not segregate with schizophrenia in families. CONCLUSIONS: Many common SNPs are likely to contribute to schizophrenia risk, with substantial overlap in genetic risk factors between multiply affected families and cases in large case-control studies. Our findings are consistent with a role for specific CNVs in disease pathogenesis, but the partial segregation of some CNVs with schizophrenia suggests that researchers should exercise caution in using them for predictive genetic testing until their effects in diverse populations have been fully studied.


Assuntos
Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Esquizofrenia/genética , População Negra/genética , Estudos de Casos e Controles , Variações do Número de Cópias de DNA/genética , Feminino , Estudo de Associação Genômica Ampla/métodos , Genótipo , Humanos , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único/genética , População Branca/genética
17.
Stem Cells Dev ; 21(4): 539-53, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21561385

RESUMO

In a perspective of regenerative medicine, multipotent human neural progenitor cells (hNPCs) offer a therapeutic advantage over pluripotent stem cells in that they are already invariantly "neurally committed" and lack tumorigenicity. However, some of their intrinsic properties, such as slow differentiation and uncontrolled multipotency, remain among the obstacles to their routine use for transplantation. Although rodent NPCs have been genetically modified in vitro to overcome some of these limitations, the translation of this strategy to human cells remains in its early stages. In the present study, we compare the actions of 4 basic helix-loop-helix transcription factors on the proliferation, specification, and terminal differentiation of hNPCs isolated from the fetal dorsal telencephalon. Consistent with their proneural activity, Ngn1, Ngn2, Ngn3, and Mash1 prompted rapid commitment of the cells. The Ngns induced a decrease in proliferation, whereas Mash1 maintained committed progenitors in a proliferative state. As opposed to Ngn1 and Ngn3, which had no effect on glial differentiation, Ngn2 induced an increase in astrocytes in addition to neurons, whereas Mash1 led to both neuronal and oligodendroglial specification. GABAergic, cholinergic, and motor neuron differentiations were considerably increased by overexpression of Ngn2 and, to a lesser extent, of Ngn3 and Mash1. Thus, we provide evidence that hNPCs can be efficiently, rapidly, and safely expanded in vitro as well as rapidly differentiated toward mature neural (typically neuronal) lineages by the overexpression of select proneural genes.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Diferenciação Celular/fisiologia , Córtex Cerebral/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios GABAérgicos/metabolismo , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Células-Tronco/metabolismo , Astrócitos/citologia , Astrócitos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Cultivadas , Córtex Cerebral/citologia , Neurônios Colinérgicos/citologia , Feto , Neurônios GABAérgicos/citologia , Regulação da Expressão Gênica/fisiologia , Humanos , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Células-Tronco/citologia
18.
Genome Med ; 3(7): 43, 2011 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-21745417

RESUMO

We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.

19.
Proc Natl Acad Sci U S A ; 108(32): 13141-6, 2011 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-21788492

RESUMO

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.


Assuntos
Eritrócitos/patologia , Eritropoese , Serotonina/deficiência , Anemia Macrocítica/complicações , Anemia Macrocítica/enzimologia , Anemia Macrocítica/patologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Suplementos Nutricionais , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Células Precursoras Eritroides/metabolismo , Células Precursoras Eritroides/patologia , Eritropoese/efeitos dos fármacos , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Serotonina/metabolismo , Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Siderose/complicações , Siderose/patologia , Baço/efeitos dos fármacos , Baço/patologia , Triptofano Hidroxilase/deficiência , Triptofano Hidroxilase/metabolismo
20.
FASEB J ; 25(9): 3271-8, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21670064

RESUMO

Transient environmental influences, such as perinatal nutritional stress, may induce deleterious metabolic symptoms that last for the entire life of individuals, implying that epigenetic modifications play an important role in this process. We have investigated, in mice, the consequences of maternal undernutrition during gestation and lactation on DNA methylation and expression of the leptin gene, which plays a major regulatory role in coordinating nutritional state with many aspects of mammalian biology. We show that animals born to mothers fed a low-protein-diet (F1-LPD group) have a lower body weight/adiposity and exhibit a higher food intake than animals born to mothers fed a control diet (F1-CD group). These modifications persisted throughout life and were associated with lower levels of leptin mRNA and protein in starved F1-LPD mice, emphasizing that maternal protein-undernutrition affects the balance between food intake and energy expenditure in adults. Moreover, this nutritional stress resulted in the removal of methyls at CpGs located in the promoter of leptin, causing a permanent specific modification in the dynamics of the expression of leptin, which exhibits a stronger induction in the F1-LPD than in F1-CD mice in response to a meal. This study is an example of a molecular rationale linking transient environmental influences to permanent phenotypic consequences.


Assuntos
Dieta , Proteínas Alimentares/farmacologia , Leptina/metabolismo , Síndrome Metabólica/etiologia , Fenômenos Fisiológicos da Nutrição Pré-Natal , Animais , Sequência de Bases , Composição Corporal , Ilhas de CpG , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Leptina/genética , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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