RESUMO
Background and aims Colonoscopy is currently the reference method to detect colorectal neoplasia, yet some adenomas remain undetected. The water infusion technique and dying with indigo carmine has shown interesting results for reducing this miss rate. The aim of this study was to compare the adenoma detection rate (adenoma and adenocarcinoma; ADR) and the mean number of adenomas per patient (MAP) for blue-water infusion colonoscopy (BWIC) versus standard colonoscopy. Methods We performed a multicenter, randomized controlled trial in eight units, including patients with a validated indication for colonoscopy (symptoms, familial or personal history, fecal occult blood test positive). Consenting patients were randomized 1:1 to BWIC or standard colonoscopy. All colonoscopies were performed by experienced colonoscopists. All colonoscopy quality indicators were prospectively recorded. Results Among the 1065 patients included, colonoscopies were performed completely for 983 patients (514 men; mean age 59.1). The ADR was not significantly different between the groups; 40.4â% in the BWIC group versus 37.5â% in the standard colonoscopy group (odds ratio [OR] 1.13; 95â% confidence interval [CI] 0.87â-â1.48; Pâ=â0.35). MAP was significantly greater in the BWIC group (0.79) than in the standard colonoscopy group (0.64; Pâ=â0.005). For advanced adenomas, the results were 50 (10.2â%) and 36 (7.3â%), respectively (Pâ=â0.10). The cecal intubation rate was not different but the time to cecal intubation was significantly longer in BWIC group (9.9 versus 6.2 minutes; Pâ<â0.001). Conclusion Despite the higher MAP with BWIC, the routine use of BWIC does not translate to a higher ADR. Whether increased detection ultimately results in a lower rate of interval carcinoma is not yet known. CLINICAL TRIALS REGISTRATION: EudraCT 2012-A00548â-â35; NCT01937429.
Assuntos
Adenocarcinoma/diagnóstico por imagem , Adenoma/diagnóstico por imagem , Colonoscopia/métodos , Neoplasias Colorretais/diagnóstico por imagem , Idoso , Ceco , Cor , Feminino , Humanos , Intubação Gastrointestinal , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , ÁguaRESUMO
The main analytical use of Ca(2+)-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca(2+)]i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated. Only analogues having electron-donating groups (m-OCH3 and m-OH) on the C6 phenol moiety or an extended resonance system at the C8 position (1-naphthyl and α-styryl analogues) showed a significant red shift of light emission. Of these, only the α-styryl analogue displayed a sufficiently high light intensity to allow eventual tissue penetration. The possible suitability of this compound for in vivo assays was corroborated by studies with aequorin which allowed the monitoring of [Ca(2+)]i dynamics in cultured CHO cells and in hippocampal brain slices. Thus, the α-styryl coelenterazine analogue might be potentially useful for non-invasive, in vivo bioluminescence imaging in deep tissues of small animals.
Assuntos
Equorina/química , Imidazóis/química , Substâncias Luminescentes/química , Medições Luminescentes/instrumentação , Proteínas Luminescentes/química , Imagem Molecular/instrumentação , Pirazinas/química , Animais , Células CHO , Cricetulus , Hidrozoários , Imagem Molecular/métodosRESUMO
The construction and application of genetically encoded intracellular calcium concentration ([Ca2+]i) indicators has a checkered history. Excitement raised over the creation of new probes is often followed by disappointment when it is found that the initial demonstrations of [Ca2+]i sensing capability cannot be leveraged into real scientific advances. Recombinant apo-aequorin cloned from Aequorea victoria was the first Ca2+ sensitive protein genetically targeted to subcellular compartments. In the jellyfish, bioluminescence resonance energy transfer (BRET) between Ca2+ bound aequorin and green fluorescent protein (GFP) emits green light. Similarly, Ca2+ sensitive bioluminescent reporters undergoing BRET have been constructed between aequorin and GFP, and more recently with other fluorescent protein variants. These hybrid proteins display red-shifted spectrums and have higher light intensities and stability compared to aequorin alone. We report BRET measurement of single-cell [Ca2+]i based on the use of electron-multiplying charge-coupled-detector (EMCCD) imaging camera technology, mounted on either a bioluminescence or conventional microscope. Our results show for the first time how these new technologies make facile long-term monitoring of [Ca2+]i at the single-cell level, obviating the need for expensive, fragile, and sophisticated equipment based on image-photon-detectors (IPD) that were until now the only technical recourse to dynamic BRET experiments of this type.
Assuntos
Equorina/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Medições Luminescentes/instrumentação , Proteínas Luminescentes/metabolismo , Técnicas de Sonda Molecular/instrumentação , Transdutores , Elétrons , Desenho de Equipamento , Análise de Falha de Equipamento , Medições Luminescentes/métodosRESUMO
The non-toxic C fragment of tetanus toxin fused to the beta-galactosidase enzyme was analysed as a neuroanatomical tracer. After intramuscular injection in rat tongue, its location in the hypoglossal network was compared with other classic tracers such as neurotropic viruses. The hybrid protein reached second and higher-order neurons after crossing several synapses. It appears to be a powerful tool to map neuronal circuits since the protein is easy to handle and detect and its transsynaptic transport is potential activity-dependent.
Assuntos
Encéfalo/anatomia & histologia , Fragmentos de Peptídeos , beta-Galactosidase , Animais , Encéfalo/citologia , Corantes , Nervo Hipoglosso/anatomia & histologia , Nervo Hipoglosso/citologia , Imuno-Histoquímica , Neurônios Motores/fisiologia , Ratos , Ratos Wistar , Toxina TetânicaRESUMO
Many different cells' signalling pathways are universally regulated by Ca(2+) concentration [Ca(2+)] rises that have highly variable amplitudes and kinetic properties. Optical imaging can provide the means to characterise both the temporal and spatial aspects of Ca(2+) signals involved in neurophysiological functions. New methods for in vivo imaging of Ca(2+) signalling in the brain of Drosophila are required for probing the different dynamic aspects of this system. In studies here, whole brain Ca(2+) imaging was performed on transgenic flies with targeted expression of the bioluminescent Ca(2+) reporter GFP-aequorin (GA) in different neural structures. A photon counting based technique was used to undertake continuous recordings of cytosolic [Ca(2+)] over hours. Time integrals for reconstructing images and analysis of the data were selected offline according to the signal intensity. This approach allowed a unique Ca(2+) response associated with cholinergic transmission to be identified by whole brain imaging of specific neural structures. Notably, [Ca(2+)] transients in the Mushroom Bodies (MBs) following nicotine stimulation were accompanied by a delayed secondary [Ca(2+)] rise (up to 15 min. later) in the MB lobes. The delayed response was sensitive to thapsigargin, suggesting a role for intra-cellular Ca(2+) stores. Moreover, it was reduced in dunce mutant flies, which are impaired in learning and memory. Bioluminescence imaging is therefore useful for studying Ca(2+) signalling pathways and for functional mapping of neurophysiological processes in the fly brain.
Assuntos
Encéfalo/fisiologia , Cálcio/fisiologia , Drosophila/fisiologia , Animais , Animais Geneticamente Modificados , Encéfalo/anatomia & histologia , Drosophila/genética , Drosophila/efeitos da radiação , Marcadores Genéticos , Proteínas de Fluorescência Verde/genética , Luz , Luminescência , Proteínas Luminescentes/genética , Proteínas Luminescentes/fisiologia , Transdução de SinaisRESUMO
Real-time visualization of calcium (Ca(2+)) dynamics in the whole animal will enable important advances in understanding the complexities of cellular function. The genetically encoded bioluminescent Ca(2+) reporter green fluorescent protein-aequorin (GA) allows noninvasive detection of intracellular Ca(2+) signaling in freely moving mice. However, the emission spectrum of GA is not optimal for detection of activity from deep tissues in the whole animal. To overcome this limitation, two new reporter genes were constructed by fusing the yellow fluorescent protein (Venus) and the monomeric red fluorescent protein (mRFP1) to aequorin. Transfer of aequorin chemiluminescence energy to Venus (VA) is highly efficient and produces a 58 nm red shift in the peak emission spectrum of aequorin. This substantially improves photon transmission through tissue, such as the skin and thoracic cage. Although the Ca(2+)-induced bioluminescence spectrum of mRFP1-aequorin (RA) is similar to that of aequorin, there is also a small peak above 600 nm corresponding to the peak emission of mRFP1. Small amounts of energy transfer between aequorin and mRFP1 yield an emission spectrum with the highest percentage of total light above 600 nm compared with GA and VA. Accordingly, RA is also detected with higher sensitivity from brain areas. VA and RA will therefore improve optical access to Ca(2+) signaling events in deeper tissues, such as the heart and brain, and offer insight for engineering new hybrid molecules.
Assuntos
Equorina/análise , Proteínas de Bactérias/análise , Sinalização do Cálcio , Genes Reporter , Substâncias Luminescentes/análise , Proteínas Luminescentes/análise , Proteínas Recombinantes de Fusão/análise , Imagem Corporal Total/métodos , Equorina/genética , Animais , Proteínas de Bactérias/genética , Transferência de Energia , Medições Luminescentes , Proteínas Luminescentes/genética , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteína Vermelha FluorescenteRESUMO
Rapid and transient elevations of Ca(2+) within cellular microdomains play a critical role in the regulation of many signal transduction pathways. Described here is a genetic approach for non-invasive detection of localized Ca(2+) concentration ([Ca(2+)]) rises in live animals using bioluminescence imaging (BLI). Transgenic mice conditionally expressing the Ca(2+)-sensitive bioluminescent reporter GFP-aequorin targeted to the mitochondrial matrix were studied in several experimental paradigms. Rapid [Ca(2+)] rises inside the mitochondrial matrix could be readily detected during single-twitch muscle contractions. Whole body patterns of [Ca(2+)] were monitored in freely moving mice and during epileptic seizures. Furthermore, variations in mitochondrial [Ca(2+)] correlated to behavioral components of the sleep/wake cycle were observed during prolonged whole body recordings of newborn mice. This non-invasive imaging technique opens new avenues for the analysis of Ca(2+) signaling whenever whole body information in freely moving animals is desired, in particular during behavioral and developmental studies.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Diagnóstico por Imagem/métodos , Animais , Animais Recém-Nascidos , Citosol/metabolismo , Diagnóstico por Imagem/instrumentação , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Contração Muscular , Neurônios/metabolismo , Oscilometria , Transdução de SinaisRESUMO
Glial Ca(2+) excitability plays a key role in reciprocal neuron-glia communication. In the retina, neuron-glia signalling is expected to be maximal in the dark, but the glial Ca(2+) signal characteristics under such conditions have not been evaluated. To address this question, we used bioluminescence imaging to monitor spontaneous Ca(2+) changes under dark conditions selectively in Müller cells, the principal retinal glial cells. By combining this imaging approach with network analysis, we demonstrate that activity in Müller cells is organized in networks of coactive cells, involving 2-16 cells located distantly and/or in clusters. We also report that spontaneous activity of small networks (2-6 Müller cells) repeat over time, sometimes in the same sequential order, revealing specific temporal dynamics. In addition, we show that networks of coactive glial cells are inhibited by TTX, indicating that ganglion and/or amacrine neuronal cells probably regulate Müller cell network properties. These results represent the first demonstration that spontaneous activity in adult Müller cells is patterned into correlated networks that display repeated sequences of coactivations over time. Furthermore, our bioluminescence technique provides a novel tool to study the dynamic characteristics of glial Ca(2+) events in the retina under dark conditions, which should greatly facilitate future investigations of retinal dark-adaptive processes.
Assuntos
Cálcio/metabolismo , Adaptação à Escuridão/fisiologia , Microscopia de Fluorescência/métodos , Neuroglia/fisiologia , Retina/citologia , Retina/fisiologia , Equorina/genética , Fatores Etários , Células Amácrinas/citologia , Células Amácrinas/fisiologia , Animais , Comunicação Celular/fisiologia , Células Cultivadas , Citoplasma/fisiologia , Genes Reporter/fisiologia , Proteínas de Fluorescência Verde/genética , Camundongos , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/fisiologiaRESUMO
In a previous study it was reported that fusion proteins composed of the atoxic C-terminal fragment of tetanus toxin (TTC) and green fluorescent protein or beta-galactosidase (GFP-TTC and beta-gal-TTC, respectively) rapidly cluster at motor nerve terminals of the mouse neuromuscular junction (NMJ). Because this traffic involves presynaptic activity, probably via the secretion of active molecules, we examined whether it is affected by brain-derived neurotrophic factor (BDNF). Quantitative confocal microscopy and a fluorimetric assay for beta-gal activity revealed that co-injecting BDNF and the fusion proteins significantly increased the kinetics and amount of the proteins' localization at the NMJ and their internalization by motor nerve terminals. The observed increases were independent of synaptic vesicle recycling because BDNF did not affect spontaneous quantal acetylcholine release. In addition, injecting anti-BDNF antibody shortly before injecting GFP-TTC, and before co-injecting GFP-TTC and BDNF, significantly reduced the fusion protein's localization at the NMJ. Co-injecting GFP-TTC with neurotrophin-4 (NT-4) or glial-derived neurotrophic factor (GDNF), but not with nerve growth factor, neurotrophin-3 or ciliary neurotrophic factor, also significantly increased the fusion protein's localization at the NMJ. Thus, TTC probes may use for their neuronal internalization endocytic pathways normally stimulated by BDNF, NT-4 and GDNF binding. Different tyrosine kinase receptors with similar signalling pathways are activated by BDNF/NT-4 and GDNF binding. Thus, activated components of these signalling pathways may be involved in the TTC probes' internalization, perhaps by facilitating localization of receptors of TTC in specific membrane microdomains or by recruiting various factors needed for internalization of TTC.
Assuntos
Transporte Axonal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Neurônios Motores/efeitos dos fármacos , Junção Neuromuscular/citologia , Fragmentos de Peptídeos/metabolismo , Toxina Tetânica/metabolismo , Animais , Anticorpos/farmacologia , Transporte Axonal/fisiologia , Fator Neurotrófico Derivado do Encéfalo/imunologia , Relação Dose-Resposta a Droga , Feminino , Fluorometria/métodos , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Microscopia Confocal/métodos , Proteínas de Neurofilamentos/metabolismo , Transporte Proteico/efeitos dos fármacos , Receptor trkB/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/fisiologia , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
The atoxic C-terminal fragment of tetanus neurotoxin or TTC fragment presents similar retrograde and transsynaptic properties to that of holotoxin. Detection of this fragment is easier when it is associated with a fluorescent marker or with beta-galactosidase activity by genetic fusion or chemical conjugation. Thus, these tracers have been used to study and analyse the synaptic connections of a neural network. In this article, we shortly review the various methods used with this aim including: injection of the fusion protein, adenovirus in vivo expression and transgenesis. Since neural activity is essential for neuronal TTC binding and internalization, the functionality of connections can be also evaluated. Moreover, modifications of the retrograde transport can be detected by using this fragment. Thus, TTC fragment is an excellent tracer to analyse the connectivity and functionality of a neural network. The TTC fragment was also soon proposed as potential therapeutic vector to transport and to deliver a biological activity or gene in a neural network. With this aim, the efficiency of a translocation domain to induce the cytosolic release of the associated activity has been evaluated. The use of the TTC fragment to target specifically a neurotrophic factor to neurons and thus avoid secondary effects has been tested with interesting results.
Assuntos
Rede Nervosa/ultraestrutura , Fragmentos de Peptídeos , Toxina Tetânica , Animais , Transporte Axonal , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Neurônios/ultraestrutura , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão , Sinapses/ultraestrutura , Toxina Tetânica/genética , beta-Galactosidase/genéticaRESUMO
Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto-fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca(2+)-sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca2+ responses in single cells. To overcome this limitation, we recently reported the bi-functional Ca2+ reporter gene, GFP-aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al., (2000) PNAS, 97, 7260-7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles and to the postsynaptic density. We demonstrate that these reporters enable 'real-time' measurements of subcellular Ca2+ changes in single mammalian neurons using bioluminescence. The high signal-to-noise ratio of these reporters is also important in that it affords the visualization of Ca2+ dynamics in cell-cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex-vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non-invasive monitoring of local Ca2+ dynamics and cellular communication in tissue or whole animal studies.
Assuntos
Sinalização do Cálcio/fisiologia , Medições Luminescentes/métodos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Equorina/análise , Equorina/genética , Animais , Células COS , Células Cultivadas , Córtex Cerebral/química , Chlorocebus aethiops , Humanos , Técnicas In Vitro , CamundongosRESUMO
The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP) and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immunogold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated in different axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos.
Assuntos
Proteínas de Fluorescência Verde/farmacocinética , Junção Neuromuscular/metabolismo , Fragmentos de Peptídeos/farmacocinética , Toxina Tetânica/farmacocinética , Animais , Transporte Axonal/fisiologia , Feminino , Técnicas In Vitro , Camundongos , Microscopia Imunoeletrônica , Neurônios Motores/metabolismo , Terminações Nervosas/metabolismo , Terminações Nervosas/ultraestrutura , Junção Neuromuscular/ultraestrutura , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
The distribution, dynamics, internalization, and retrograde axonal traffic of a fusion protein composed of green fluorescent protein (GFP)and the atoxic C-terminal fragment of tetanus toxin (TTC) were studied after its in vivo injection. Confocal microscopy and immuno-gold electron microscopy revealed that the fusion protein (GFP-TTC) rapidly clustered in motor nerve terminals of the neuromuscular junction. Clathrin-coated pits, and axolemma infoldings located between active zones appeared to be involved in the internalization of the fusion protein. Biochemical analysis of detergent-extracted neuromuscular preparations showed that the GFP-TTC fusion protein was associated with lipid microdomains. We suggest that GFP-TTC clustering in these lipid microdomains favors the recruitment of other proteins involved in its endocytosis and internalization in motor nerve terminals. During its retrograde trafficking, GFP-TTC accumulated indifferent axonal compartments than those used by cholera toxin B-subunit suggesting that these two proteins are transported by different pathways and cargos.
Assuntos
Transporte Axonal/fisiologia , Endocitose/fisiologia , Junção Neuromuscular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Membranas Sinápticas/metabolismo , Animais , Toxina da Cólera/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/ultraestrutura , Feminino , Proteínas de Fluorescência Verde/metabolismo , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/ultraestrutura , Camundongos , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Junção Neuromuscular/ultraestrutura , Fragmentos de Peptídeos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Transporte Proteico/fisiologia , Membranas Sinápticas/ultraestrutura , Toxina Tetânica/metabolismo , Tomografia por Raios XRESUMO
Retinoic acid (RA) activity plays sequential roles during the development of the ventral spinal cord. Here, we have investigated the functions of local RA synthesis in the process of motoneuron specification and early differentiation using a conditional knockout strategy that ablates the function of the retinaldehyde dehydrogenase 2 (Raldh2) synthesizing enzyme essentially in brachial motoneurons, and later in mesenchymal cells at the base of the forelimb. Mutant (Raldh2L-/-) embryos display an early embryonic loss of a subset of Lim1+ brachial motoneurons, a mispositioning of Islet1+ neurons and inappropriate axonal projections of one of the nerves innervating extensor limb muscles, which lead to an adult forepaw neuromuscular defect. The molecular basis of the Raldh2L-/- phenotype relies in part on the deregulation of Hoxc8, which in turn regulates the RA receptor RARbeta. We further show that Hoxc8 mutant mice, which exhibit a similar congenital forepaw defect, display at embryonic stages molecular defects that phenocopy the Raldh2L-/- motoneuron abnormalities. Thus, interdependent RA signaling and Hox gene functions are required for the specification of brachial motoneurons in the mouse.
Assuntos
Aldeído Oxirredutases/genética , Proteínas de Homeodomínio/genética , Neurônios Motores/metabolismo , Medula Espinal/embriologia , Aldeído Oxirredutases/metabolismo , Animais , Proteínas de Homeodomínio/metabolismo , Proteínas com Homeodomínio LIM , Camundongos , Camundongos Knockout , Mutação , Medula Espinal/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The function of the nervous system is a consequence of the intricate synaptic connectivity of its neurons. Our understanding of these highly complex networks has profited enormously from methods used over the past two decades that are based on the mechanical injection of tracer molecules into brain regions. We have developed a genetic system for the mapping of synaptic connections during development of the mammalian central nervous system and in the mature brain. It is based on the transsynaptic transfer of green fluorescent protein (GFP) in the brains of mice using a fusion protein with a nontoxic fragment of tetanus toxin (TTC) expressed in defined neurons. These transgenic mice allowed us to visualize neurons, at single-cell resolution, that are in synaptic contact by the detection of GFP in interconnected circuits. Targeted genetic expression with a specific promoter permitted us to transfer GFP to defined subsets of neurons and brain regions. GFP-TTC is coexpressed with a lacZ reporter gene to discriminate neurons that produce the tracer from cells that have acquired it transneuronally. The marker shows selective transfer in the retrograde direction. We have used electron microscopic detection of GFP to define the ultrastructural features of the system. Our work opens up a range of possibilities for brain slice and in vivo studies taking advantage of the fluorescence of GFP. We point the way toward the use of powerful multiphoton technology and set the stage for the transsynaptic transfer of other proteins in the brains of mice.
Assuntos
Mapeamento Cromossômico , Proteínas Luminescentes/genética , Neurônios/fisiologia , Sinapses/fisiologia , Animais , Sequência de Bases , Primers do DNA , Genes Reporter , Proteínas de Fluorescência Verde , Hibridização In Situ , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
During development and also in adulthood, synaptic connections are modulated by neuronal activity. To follow such modifications in vivo, new genetic tools are designed. The nontoxic C-terminal fragment of tetanus toxin (TTC) fused to a reporter gene such as LacZ retains the retrograde and transsynaptic transport abilities of the holotoxin itself. In this work, the hybrid protein is injected intramuscularly to analyze in vivo the mechanisms of intracellular and transneuronal traffics at the neuromuscular junction (NMJ). Traffic on both sides of the synapse are strongly dependent on presynaptic neural cell activity. In muscle, a directional membrane traffic concentrates beta-galactosidase-TTC hybrid protein into the NMJ postsynaptic side. In neurons, the probe is sorted across the cell to dendrites and subsequently to an interconnected neuron. Such fusion protein, sensitive to presynaptic neuronal activity, would be extremely useful to analyze morphological changes and plasticity at the NMJ.
Assuntos
Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Animais , Membrana Celular/metabolismo , Endocitose , Genes Reporter , Cinética , Camundongos , Neurônios/fisiologia , Fragmentos de Peptídeos/metabolismo , Toxina Tetânica/metabolismo , beta-Galactosidase/metabolismoRESUMO
Gene therapy vectors that can be targeted to motoneuronal cells are required in the field of neurodegenerative diseases. We propose the use of the atoxic fragment C of tetanus toxin (TTC) as biological activity carrier to the motoneurons. Naked DNA encoding beta-galactosidase-TTC hybrid protein was used to transfect muscle cells in vivo, resulting in a selective gene transfer of the enzymatic activity to the CNS. In the muscle, level expression of beta-galactosidase was readily detectable 24 h after injection, reaching a maximum after 4 days and gradually decreasing thereafter. Labelling in the hypoglossal motoneurons and motor cortex was observed from 4 days after injection. In this paper, we show that TTC works as an enzymatic activity carrier to the CNS when muscle cells are transfected in vivo. We have also shown that the presence of TTC does not have any influence on the expression of the transfected gene. Both these results warrant further studies of TTC as a means of treating motoneuron diseases in the field of gene therapy.
Assuntos
Sistema Nervoso Central/citologia , Terapia Genética/métodos , Vetores Genéticos/genética , Neurônios Motores/fisiologia , Fragmentos de Peptídeos/farmacologia , Toxina Tetânica/farmacologia , Animais , Sistema Nervoso Central/ultraestrutura , DNA/genética , DNA/farmacologia , Relação Dose-Resposta a Droga , Genes Reporter/genética , Vetores Genéticos/metabolismo , Camundongos , Microscopia Eletrônica , Neurônios Motores/ultraestrutura , Músculos/inervação , Músculos/metabolismo , Fragmentos de Peptídeos/metabolismo , Plasmídeos/genética , Ratos , Ratos Wistar , Toxina Tetânica/metabolismo , beta-Galactosidase/genéticaRESUMO
The retrograde transport and transynaptic transfer properties of the nontoxic tetanus toxin C-fragment (TTC) can be used to visualize specific neural pathways or to deliver biomolecules in the central nervous system (CNS). Here we tested different delivery techniques to explore the potential use of a new GFP-TTC fusion construct for use as a genetic tracer in vivo. Plasmids encoding GFP-TTC were targeted to brain regions using intracerebral grafted transfected cells or adenoviral transduction. Transport was monitored using GFP fluorescence. We show that following GFP-TTC synthesis in grafted transfected cells, the TTC fragment alone, with no signal peptide, is necessary and sufficient to provide secretion and uptake of the fusion protein into neighboring neurons around the injection site. Using an adenoviral vector to express the fusion protein into brain neurons, we show that transduced neurons can deliver the fusion protein specifically into connected neurons, demonstrating that synaptic transfer in the CNS can be visualized with GFP-TTC.