RESUMO
Macroalgae provide key contributions to aquatic ecosystems, including their role as primary producers and the provision of biotopes for marine organisms, fish spawning, and fish nurseries. The aim of this study was to evaluate the feasibility of a micronucleus test and a comet assay in Ceramium tenuicorne, a red macroalga. This alga is widely distributed in marine ecosystems and brackish waters, and is therefore a potential bioindicator of the effects of anthropogenic pollution in these ecosystems. Unfortunately, the two genotoxicity tests evaluated were not suitable for this alga because the nuclei are generally very small (between 2 and 10 µm), are variable in size, and there may be several nuclei in each cell (between 1 and 5 in our observations). However, the present study allowed us to define conditions for observing these algal cells and optimizing the choice of DNA dye (orcein), cell fixation solution (Carnoy's solution), and hydrolysis step (HCl, 1200 mmol/L solution for 1 min). This research allowed us to propose two genotoxicity and cytotoxicity endpoints for assessing chemical effects on the algal cells: counting of nuclei in cortical cell areas, and the frequency of axial cells in mitosis. These two criteria were measured after exposing C. tenuicorne to two reference substances: cadmium chloride and maleic hydrazide, and we highlight the effects from 1 × 10-5 M of CdCl2 and 5 × 10-5 M of maleic hydrazide.
RESUMO
In mammals, including humans, nearly all physiological processes are subject to daily oscillations that are governed by a circadian timing system with a complex hierarchical structure. The central pacemaker, residing in the suprachiasmatic nucleus (SCN) of the ventral hypothalamus, is synchronized daily by photic cues transmitted from the retina to SCN neurons via the retinohypothalamic tract. In turn, the SCN must establish phase coherence between self-sustained and cell-autonomous oscillators present in most peripheral cell types. The synchronization signals (Zeitgebers) can be controlled more or less directly by the SCN. In mice and rats, feeding-fasting rhythms, which are driven by the SCN through rest-activity cycles, are the most potent Zeitgebers for the circadian oscillators of peripheral organs. Signaling through the glucocorticoid receptor and the serum response factor also participate in the phase entrainment of peripheral clocks, and these two pathways are controlled by the SCN independently of feeding-fasting rhythms. Body temperature rhythms, governed by the SCN directly and indirectly through rest-activity cycles, are perhaps the most surprising cues for peripheral oscillators. Although the molecular makeup of circadian oscillators is nearly identical in all cells, these oscillators are used for different purposes in the SCN and in peripheral organs.
Assuntos
Actinas/metabolismo , Temperatura Corporal/fisiologia , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Retina/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Relógios Biológicos , Sinais (Psicologia) , Jejum/fisiologia , Comportamento Alimentar/fisiologia , Humanos , Mamíferos , Camundongos , Ratos , Transdução de SinaisRESUMO
STUDY OBJECTIVES: That sleep deprivation increases the brain expression of various clock genes has been well documented. Based on these and other findings we hypothesized that clock genes not only underlie circadian rhythm generation but are also implicated in sleep homeostasis. However, long time lags have been reported between the changes in the clock gene messenger RNA levels and their encoded proteins. It is therefore crucial to establish whether also protein levels increase within the time frame known to activate a homeostatic sleep response. We report on the central and peripheral effects of sleep deprivation on PERIOD-2 (PER2) protein both in intact and suprachiasmatic nuclei-lesioned mice. DESIGN: In vivo and in situ PER2 imaging during baseline, sleep deprivation, and recovery. SETTINGS: Mouse sleep-recording facility. PARTICIPANTS: Per2::Luciferase knock-in mice. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Six-hour sleep deprivation increased PER2 not only in the brain but also in liver and kidney. Remarkably, the effects in the liver outlasted those observed in the brain. Within the brain the increase in PER2 concerned the cerebral cortex mainly, while leaving suprachiasmatic nuclei (SCN) levels unaffected. Against expectation, sleep deprivation did not increase PER2 in the brain of arrhythmic SCN-lesioned mice because of higher PER2 levels in baseline. In contrast, liver PER2 levels did increase in these mice similar to the sham and partially lesioned controls. CONCLUSIONS: Our results stress the importance of considering both sleep-wake dependent and circadian processes when quantifying clock-gene levels. Because sleep deprivation alters PERIOD-2 in the brain as well as in the periphery, it is tempting to speculate that clock genes constitute a common pathway mediating the shared and well-known adverse effects of both chronic sleep loss and disrupted circadian rhythmicity on metabolic health.
Assuntos
Ritmo Circadiano/fisiologia , Homeostase/fisiologia , Proteínas Circadianas Period/metabolismo , Privação do Sono/fisiopatologia , Sono/fisiologia , Núcleo Supraquiasmático/fisiopatologia , Animais , Encéfalo/metabolismo , Feminino , Técnicas de Introdução de Genes , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Circadianas Period/genética , Núcleo Supraquiasmático/lesões , Vigília/fisiologiaRESUMO
The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano , Regulação da Expressão Gênica , Hepatócitos/fisiologia , Fígado/metabolismo , Atividade Motora/fisiologia , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Comportamento Alimentar , Fígado/citologia , Medições Luminescentes , Masculino , Camundongos , Camundongos Pelados , Atividade Motora/genética , Transdução de Sinais , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/cirurgiaRESUMO
Maintaining wakefulness is associated with a progressive increase in the need for sleep. This phenomenon has been linked to changes in synaptic function. The synaptic adhesion molecule Neuroligin-1 (NLG1) controls the activity and synaptic localization of N-methyl-d-aspartate receptors, which activity is impaired by prolonged wakefulness. We here highlight that this pathway may underlie both the adverse effects of sleep loss on cognition and the subsequent changes in cortical synchrony. We found that the expression of specific Nlg1 transcript variants is changed by sleep deprivation in three mouse strains. These observations were associated with strain-specific changes in synaptic NLG1 protein content. Importantly, we showed that Nlg1 knockout mice are not able to sustain wakefulness and spend more time in nonrapid eye movement sleep than wild-type mice. These changes occurred with modifications in waking quality as exemplified by low theta/alpha activity during wakefulness and poor preference for social novelty, as well as altered delta synchrony during sleep. Finally, we identified a transcriptional pathway that could underlie the sleep/wake-dependent changes in Nlg1 expression and that involves clock transcription factors. We thus suggest that NLG1 is an element that contributes to the coupling of neuronal activity to sleep/wake regulation.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Neurônios/fisiologia , Sono/fisiologia , Vigília/fisiologia , Animais , Western Blotting , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Eletroencefalografia , Eletromiografia , Expressão Gênica , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Neurônios/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sono/genética , Privação do Sono/genética , Privação do Sono/fisiopatologia , Especificidade da Espécie , Fatores de Tempo , Vigília/genéticaRESUMO
STUDY OBJECTIVES: Besides their well-established role in circadian rhythms, our findings that the forebrain expression of the clock-genes Per2 and Dbp increases and decreases, respectively, in relation to time spent awake suggest they also play a role in the homeostatic aspect of sleep regulation. Here, we determined whether time of day modulates the effects of elevated sleep pressure on clock-gene expression. Time of day effects were assessed also for recognized electrophysiological (EEG delta power) and molecular (Homer1a) markers of sleep homeostasis. DESIGN: EEG and qPCR data were obtained for baseline and recovery from 6-h sleep deprivation starting at ZT0, -6, -12, or -18. SETTING: Mouse sleep laboratory. PARTICIPANTS: Male mice. INTERVENTIONS: Sleep deprivation. RESULTS: The sleep-deprivation induced changes in Per2 and Dbp expression importantly varied with time of day, such that Per2 could even decrease during sleep deprivations occurring at the decreasing phase in baseline. Dbp showed similar, albeit opposite dynamics. These unexpected results could be reliably predicted assuming that these transcripts behave according to a driven damped harmonic oscillator. As expected, the sleep-wake distribution accounted for a large degree of the changes in EEG delta power and Homer1a. Nevertheless, the sleep deprivation-induced increase in delta power varied also with time of day with higher than expected levels when recovery sleep started at dark onset. CONCLUSIONS: Per2 and delta power are widely used as exclusive state variables of the circadian and homeostatic process, respectively. Our findings demonstrate a considerable cross-talk between these two processes. As Per2 in the brain responds to both sleep loss and time of day, this molecule is well positioned to keep track of and to anticipate homeostatic sleep need. CITATION: Curie T; Mongrain V; Dorsaz S; Mang GM; Emmenegger Y; Franken P. Homeostatic and circadian contribution to EEG and molecular state variables of sleep regulation. SLEEP 2013;36(3):311-323.
Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Ligação a DNA/metabolismo , Eletroencefalografia/métodos , Homeostase/fisiologia , Proteínas Circadianas Period/metabolismo , Sono/fisiologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Expressão Gênica/fisiologia , Proteínas de Arcabouço Homer , Masculino , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos C57BL , Prosencéfalo/metabolismo , Privação do Sono/metabolismoRESUMO
Although sleep is defined as a behavioral state, at the cortical level sleep has local and use-dependent features suggesting that it is a property of neuronal assemblies requiring sleep in function of the activation experienced during prior wakefulness. Here we show that mature cortical cultured neurons display a default state characterized by synchronized burst-pause firing activity reminiscent of sleep. This default sleep-like state can be changed to transient tonic firing reminiscent of wakefulness when cultures are stimulated with a mixture of waking neurotransmitters and spontaneously returns to sleep-like state. In addition to electrophysiological similarities, the transcriptome of stimulated cultures strikingly resembles the cortical transcriptome of sleep-deprived mice, and plastic changes as reflected by AMPA receptors phosphorylation are also similar. We used our in vitro model and sleep-deprived animals to map the metabolic pathways activated by waking. Only a few metabolic pathways were identified, including glycolysis, aminoacid, and lipids. Unexpectedly large increases in lysolipids were found both in vivo after sleep deprivation and in vitro after stimulation, strongly suggesting that sleep might play a major role in reestablishing the neuronal membrane homeostasis. With our in vitro model, the cellular and molecular consequences of sleep and wakefulness can now be investigated in a dish.
Assuntos
Potenciais de Ação/fisiologia , Córtex Cerebral , Sono/fisiologia , Vigília/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/química , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Privação do Sono/metabolismoRESUMO
Although the pathology of Morbillivirus in the central nervous system (CNS) is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV) that we inoculated into two different cell systems: a monkey cell line (Vero) and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H) markedly accumulated in the endoplasmic reticulum (ER). This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT), another ER resident chaperon critically involved in the response to misfolded proteins and in Ca(2+) homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca(2+) homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses.
Assuntos
Cálcio/metabolismo , Calreticulina/metabolismo , Vírus da Cinomose Canina/genética , Estresse do Retículo Endoplasmático , Glicoproteínas/genética , Homeostase , Fragmentos de Peptídeos/metabolismo , Proteínas Virais/genética , Animais , Membrana Celular/metabolismo , Chlorocebus aethiops , Vírus da Cinomose Canina/fisiologia , Expressão Gênica , Hipocampo/citologia , Neurônios/metabolismo , Neurônios/virologia , Transporte Proteico , Ratos , Regulação para Cima , Células VeroRESUMO
We have previously demonstrated that clock genes contribute to the homeostatic aspect of sleep regulation. Indeed, mutations in some clock genes modify the markers of sleep homeostasis and an increase in homeostatic sleep drive alters clock gene expression in the forebrain. Here, we investigate a possible mechanism by which sleep deprivation (SD) could alter clock gene expression by quantifying DNA-binding of the core-clock transcription factors CLOCK, NPAS2, and BMAL1 to the cis-regulatory sequences of target clock genes in mice. Using chromatin immunoprecipitation (ChIP), we first showed that, as reported for the liver, DNA-binding of CLOCK and BMAL1 to target clock genes changes in function of time-of-day in the cerebral cortex. Tissue extracts were collected at ZT0 (light onset), -6, -12, and -18, and DNA enrichment of E-box or E'-box containing sequences was measured by qPCR. CLOCK and BMAL1 binding to Cry1, Dbp, Per1, and Per2 depended on time-of-day, with maximum values reached at around ZT6. We then observed that SD, performed between ZT0 and -6, significantly decreased DNA-binding of CLOCK and BMAL1 to Dbp, consistent with the observed decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was also decreased by SD, although SD is known to increase Per2 expression in the cortex. DNA-binding to Per1 and Cry1 was not affected by SD. Our results show that the sleep-wake history can affect the clock molecular machinery directly at the level of chromatin binding thereby altering the cortical expression of Dbp and Per2 and likely other targets. Although the precise dynamics of the relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive, the results also suggest that part of the reported circadian changes in DNA-binding of core clock components in tissues peripheral to the suprachiasmatic nuclei could, in fact, be sleep-wake driven.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas CLOCK/metabolismo , Córtex Cerebral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Privação do Sono/metabolismo , Fatores de Transcrição ARNTL/genética , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas CLOCK/genética , Imunoprecipitação da Cromatina , Primers do DNA , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase , Ligação Proteica , Privação do Sono/genética , Ativação TranscricionalRESUMO
STUDY OBJECTIVES: The sleep-deprivation-induced changes in delta power, an electroencephalographical correlate of sleep need, and brain transcriptome profiles have importantly contributed to current hypotheses on sleep function. Because sleep deprivation also induces stress, we here determined the contribution of the corticosterone component of the stress response to the electrophysiological and molecular markers of sleep need in mice. DESIGN: N/A SETTINGS: Mouse sleep facility. PARTICIPANTS: C57BL/6J, AKR/J, DBA/2J mice. INTERVENTIONS: Sleep deprivation, adrenalectomy (ADX). MEASUREMENTS AND RESULTS: Sleep deprivation elevated corticosterone levels in 3 inbred strains, but this increase was larger in DBA/2J mice; i.e., the strain for which the rebound in delta power after sleep deprivation failed to reach significance. Elimination of the sleep-deprivation-associated corticosterone surge through ADX in DBA/2J mice did not, however, rescue the delta power rebound but did greatly reduce the number of transcripts affected by sleep deprivation. Genes no longer affected by sleep deprivation cover pathways previously implicated in sleep homeostasis, such as lipid, cholesterol (e.g., Ldlr, Hmgcs1, Dhcr7, -24, Fkbp5), energy and carbohydrate metabolism (e.g., Eno3, G6pc3, Mpdu1, Ugdh, Man1b1), protein biosynthesis (e.g., Sgk1, Alad, Fads3, Eif2c2, -3, Mat2a), and some circadian genes (Per1, -3), whereas others, such as Homer1a, remained unchanged. Moreover, several microRNAs were affected both by sleep deprivation and ADX. CONCLUSIONS: Our findings indicate that corticosterone contributes to the sleep-deprivation-induced changes in brain transcriptome that have been attributed to wakefulness per se. The study identified 78 transcripts that respond to sleep loss independent of corticosterone and time of day, among which genes involved in neuroprotection prominently feature, pointing to a molecular pathway directly relevant for sleep function.
Assuntos
Glucocorticoides/fisiologia , Homeostase/fisiologia , Privação do Sono/etiologia , Vigília/fisiologia , Adrenalectomia , Animais , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Corticosterona/metabolismo , Modelos Animais de Doenças , Eletroencefalografia , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos , RNA Mensageiro/metabolismo , Privação do Sono/metabolismo , Privação do Sono/fisiopatologiaRESUMO
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
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
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.