RESUMEN
Histological processing of thermosensitive electrospun poly(ε-caprolactone)/poly(L-lactide) (PCL/PLA) scaffolds fails, as poly(ε-caprolactone) (PCL) is characterized by its low-melting temperature (Tm = 60 °C). Here, we present an optimized low-temperature preparation method for the histological processing of un-/cellularized thermosensitive PCL/PLA scaffolds.Our study is aimed at the establishment of an optimized dehydration and low-melting-point paraffin-embedding method of electrospun PCL/PLA scaffolds (un-/cellularized). Furthermore, we compared this method with (a) automatized dehydration and standard paraffin embedding, (b) gelatin embedding followed by automatized dehydration and standard paraffin embedding, (c) cryofixation, and (d) acrylic resin embedding methods. We investigated pepsin and proteinase K antigen retrieval for their efficiency in epitope demasking at low temperatures and evaluated protocols for immunohistochemistry and immunofluorescence for cytokeratin 7 (CK7) and in situ padlock probe technology for beta actin (ACTB). Optimized dehydration and low-melting-point paraffin embedding preserved the PCL/PLA scaffold, as the diameter and structure of its fibers were unchanged. Cells attached to the PCL/PLA scaffolds showed limited alterations in size and morphology compared to control. Epitope demasking by enzymatic pepsin digestion and immunostaining of CK7 displayed an invasion of attached cells into the scaffold. Expression of ACTB and CK7 was shown by a combination of mRNA-based in situ padlock probe technology and immunofluorescence. In contrast, gelatin stabilization followed by standard paraffin embedding led to an overall shrinkage and melting of fibers, and therefore, no further analysis was possible. Acrylic resin embedding and cyrofixation caused fiber structures that were nearly unchanged in size and diameter. However, acrylic resin-embedded scaffolds are limited to 3 µm sections, whereas cyrofixation led to a reduction of the cell size by 14% compared to low-melting paraffin embedding. The combination of low-melting-point paraffin embedding and pepsin digestion as an antigen retrieval method offers a successful opportunity for histological investigations in thermosensitive specimens.
Asunto(s)
Adhesión en Parafina , Poliésteres/química , Temperatura de Transición , Células Cultivadas , Gelatina/análisis , Humanos , Queratina-7/análisisRESUMEN
Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.
Asunto(s)
Cognición/fisiología , Hipocampo/citología , Potenciación a Largo Plazo/efectos de los fármacos , Moléculas de Adhesión de Célula Nerviosa/farmacología , Neuronas/efectos de los fármacos , Receptores AMPA/metabolismo , Transmisión Sináptica/efectos de los fármacos , Análisis de Varianza , Animales , Western Blotting , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Ensayo de Inmunoadsorción Enzimática , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Microscopía Electrónica , Microscopía Fluorescente , Neuronas/fisiología , Técnicas de Placa-Clamp , Fosforilación , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
BACKGROUND: Interleukin 1 (IL-1) is implicated in neuroinflammation, an essential component of neurodegeneration. We evaluated the potential anti-inflammatory effect of a novel peptide antagonist of IL-1 signaling, Ilantide. METHODS: We investigated the binding of Ilantide to IL-1 receptor type I (IL-1RI) using surface plasmon resonance, the inhibition of Il-1ß-induced activation of nuclear factor κB (NF-κB) in HEK-Blue cells that contained an IL-1ß-sensitive reporter, the secretion of TNF-α in macrophages, protection against IL-1-induced apoptosis in neonatal pancreatic islets, and the penetration of Ilantide through the blood-brain barrier using competitive enzyme-linked immunosorbent assay (ELISA). We studied the effects of the peptide on social behavior and memory in rat models of lipopolysaccharide (LPS)- and amyloid-induced neuroinflammation, respectively, and its effect in a rat model of experimental autoimmune enchephalomyelitis. RESULTS: Ilantide bound IL-1RI, inhibited the IL-1ß-induced activation of NF-κB, and inhibited the secretion of TNF-α in vitro. Ilantide protected pancreatic islets from apoptosis in vitro and reduced inflammation in an animal model of arthritis. The peptide penetrated the blood-brain barrier. It reduced the deficits in social activity and memory in LPS- and amyloid-treated animals and delayed the development of experimental autoimmune enchephalomyelitis. CONCLUSIONS: These findings indicate that Ilantide is a novel and potent IL-1RI antagonist that is able to reduce inflammatory damage in the central nervous system and pancreatic islets.
Asunto(s)
Antiinflamatorios/uso terapéutico , Artritis/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Animales , Animales Recién Nacidos , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Artritis/inducido químicamente , Células Cultivadas , Cerebelo/citología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inducido químicamente , Humanos , Proteína Antagonista del Receptor de Interleucina 1/química , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Lipopolisacáridos , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Wistar , Conducta Social , Transfección , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Nectins belong to a family of immunoglobulin (Ig)-like cell-adhesion molecules comprising four members, nectin-1 through nectin-4. Nectins are involved in formation of the mechanical adhesive puncta adherentia junctions of synapses. Nectins share the same overall structural topology with an extracellular region containing three Ig modules, a transmembrane region, and a cytoplasmic region. In nectin-1, the first and second Ig module in the extracellular region are necessary for the trans-interaction with nectin-3 and formation of cis-dimers, respectively. The function of the third Ig module of nectin-1 remains unknown. We here report the structure in solution of the third, membrane-proximal Ig module of mouse nectin-1 (nectin-1 Ig3) solved by means of nuclear magnetic resonance (NMR) spectroscopy. It belongs to the C1 set of the Ig superfamily. Nectin-1 Ig3 was produced as a recombinant protein and induced neurite outgrowth in primary cultures of hippocampal and cerebellar granule neurons, an effect abolished by treatment with the fibroblast growth factor receptor (FGFR) inhibitor SU5402, or by transfection with a dominant-negative FGFR1 construct. We showed by surface plasmon resonance (SPR) analysis that nectin-1 Ig3 directly interacted with various isoforms of FGFR. Nectin-1 Ig3 induced phosphorylation of FGFR1c in the same manner as the whole nectin-1 ectodomain, and promoted survival of cerebellar granule neurons induced to undergo apoptosis. Finally, we constructed a peptide, nectide, by employing in silico modeling of various FGFR ligand-binding sites. Nectide mimicked all the effects of nectin-1 Ig3. We suggest that FGFR is a downstream signaling partner of nectin-1.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Supervivencia Celular , Cristalografía por Rayos X , Factor 2 de Crecimiento de Fibroblastos/fisiología , Células HEK293 , Hipocampo/citología , Humanos , Ratones , Datos de Secuencia Molecular , Nectinas , Neuritas/metabolismo , Neuritas/fisiología , Neuronas/citología , Neuronas/fisiología , Fosforilación , Cultivo Primario de Células , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Receptores del Factor de Crecimiento Derivado de Plaquetas/química , Transducción de Señal , Resonancia por Plasmón de SuperficieRESUMEN
We recently found that S100A4, a member of the multifunctional S100 protein family, protects neurons in the injured brain and identified two sequence motifs in S100A4 mediating its neurotrophic effect. Synthetic peptides encompassing these motifs stimulated neuritogenesis and survival in vitro and mimicked the S100A4-induced neuroprotection in brain trauma. Here, we investigated a possible function of S100A4 and its mimetics in the pathologies of the peripheral nervous system (PNS). We found that S100A4 was expressed in the injured PNS and that its peptide mimetic (H3) affected the regeneration and survival of myelinated axons. H3 accelerated electrophysiological, behavioral and morphological recovery after sciatic nerve crush while transiently delaying regeneration after sciatic nerve transection and repair. On the basis of the finding that both S100A4 and H3 increased neurite branching in vitro, these effects were attributed to the modulatory effect of H3 on initial axonal sprouting. In contrast to the modest effect of H3 on the time course of regeneration, H3 had a long-term neuroprotective effect in the myelin protein P0 null mice, a model of dysmyelinating neuropathy (Charcot-Marie-Tooth type 1 disease), where the peptide attenuated the deterioration of nerve conduction, demyelination and axonal loss. From these results, S100A4 mimetics emerge as a possible means to enhance axonal sprouting and survival, especially in the context of demyelinating neuropathies with secondary axonal loss, such as Charcot-Marie-Tooth type 1 disease. Moreover, our data suggest that S100A4 is a neuroprotectant in PNS and that other S100 proteins, sharing high homology in the H3 motif, may have important functions in PNS pathologies.
Asunto(s)
Regeneración Nerviosa/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Péptidos/farmacología , Proteínas S100/farmacología , Nervio Ciático/efectos de los fármacos , Animales , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/tratamiento farmacológico , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Hipocampo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína P0 de la Mielina/genética , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fármacos Neuroprotectores/uso terapéutico , Péptidos/uso terapéutico , Ratas , Ratas Wistar , Proteínas S100/uso terapéutico , Nervio Ciático/lesiones , Nervio Ciático/fisiopatología , Nervio Tibial/efectos de los fármacos , Nervio Tibial/fisiopatologíaRESUMEN
Interleukin-4 (IL-4) is a potent antiinflammatory cytokine. However its use in the clinic is hampered by side effects. We here describe the identification of a novel synthetic peptide, termed Ph8, derived from α-helix C of IL-4, which interacts with IL-4 receptor α (IL-4Rα). Employing various cultured genetically engineered cell lines and primary lymphocytes, surface plasmon resonance, qPCR, ELISA and immunoblotting techniques we found that Ph8 bound IL-4Rα and mimicked the anti-inflammatory effects of IL-4 by inhibiting TNF-α production by macrophages in vitro. It induced phosphorylation of STAT6 65kD but inhibited phosphorylation of STAT6 110 kD induced by IL-4 in a B-cell line that expressed the type I receptor. It also inhibited the IL-4-stimulated expression of a STAT6-inducible reporter gene in cells that expressed the type II receptor. Ph8 inhibited the proliferation of Th1/2 cells and downregulated the production of IFN-γ in stimulated Th1 cells. Moreover, Ph8 did not induce any shift in Th1/Th2 profile. This is a favorable effect and it is indicating that Ph8 could block general T cell activation and inflammatory responses without further inducing the side effects generally associated with IL-4 signaling. These data collectively show that Ph8 is only a partial agonist of IL-4 mimicking its desirable properties. In agreement, Ph8 treatment of rats with collagen-induced arthritis, a Th1- and antibody- mediated disease of joint, delayed the manifestation of chronic inflammation and reduced acute inflammation in carrageenan-induced edema. Our findings indicate that Ph8 is a promising potential drug candidate for the treatment of inflammatory diseases.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Subunidad alfa del Receptor de Interleucina-4/metabolismo , Interleucina-4/farmacología , Fragmentos de Péptidos/farmacología , Animales , Artritis Experimental/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Edema/tratamiento farmacológico , Células HEK293 , Humanos , Interferón gamma/metabolismo , Interleucina-4/análogos & derivados , Interleucina-4/química , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/química , Fosforilación/efectos de los fármacos , Unión Proteica , Ratas , Ratas Wistar , Factor de Transcripción STAT6/metabolismo , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Neurexin-1 (NRXN1) and neuroligin-1 (NLGN1) are synaptic cell adhesion molecules that connect pre- and postsynaptic neurons at synapses and mediate signaling across the synapse, which modulates synaptic activity and determines the properties of neuronal networks. Defects in the genes encoding NLGN1 have been linked to cognitive diseases such as autism. The roles of both NRXN1 and NLGN1 during synaptogenesis have been studied extensively, but little is known about the role of these molecules in neuritogenesis, which eventually results in neuronal circuitry formation. The present study investigated the neuritogenic effect of NLGN1 in cultures of hippocampal neurons. Our results show that NLGN1, both in soluble and membrane-bound forms, induces neurite outgrowth that depends on the interaction with NRXN1ß and on activation of fibroblast growth factor receptor-1. In addition, we demonstrate that a synthetic peptide, termed neurolide, which is modeled after a part of the binding interface of NLGN1 for NRXN1ß, can bind to NRXN1ß and mimic the biological properties of NLGN1 in vitro.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Moléculas de Adhesión Celular , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular , Células Cultivadas , Hipocampo/citología , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Neuritas/metabolismo , Neuronas/metabolismo , Ratas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Resonancia por Plasmón de SuperficieRESUMEN
ErbB receptors not only function in cancer, but are also key developmental regulators in the nervous system. We previously identified an ErbB1 peptide antagonist, Inherbin3, that is capable of inhibiting tumor growth in vitro and in vivo. In this study, we found that inhibition of ErbB1 kinase activity and activation of ErbB4 by NRG-1ß induced neurite extension, suggesting that ErbB1 and ErbB4 act as negative and positive regulators, respectively, of the neuritogenic response. Inherbin3, inhibited activation not only of ErbB1 but also of ErbB4 in primary neurons, strongly induced neurite outgrowth in rat cerebellar granule neurons, indicating that this effect mainly was due to inhibition of ErbB1 activation.
Asunto(s)
Cerebelo/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Neuritas/efectos de los fármacos , Péptidos/farmacología , Animales , Secuencia de Bases , Células Cultivadas , Cerebelo/citología , Cartilla de ADN , Receptores ErbB/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Fosforilación , Reacción en Cadena de la Polimerasa , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Morphology and behaviour of third instar larvae of the Holomediterranean antlion species Myrmecaelurus trigranunus (Pallas) are described. Larvae are facultative pit-builders, they either ambush their prey at the surface, or dig pitfall traps that prey fall in to. Dark brown spots on dorsal and ventral sides of the head and on dorsal side of the thorax are characteristic of the larvae. Eye tubercles are not prominent. Jaws are equipped with long bristles, campaniform sensilla, sensilla coeloconica, and digitiform sensilla. A unique feature is the shape of the tips of all three teeth that is screw-like with a polyhedral surface. The body surface is covered with longitudinally grooved bristles and plumose hairs. On the tip of the antennae and on terminal and subterminal parts of labial palps sensilla basiconica occur. On the 9th abdominal segment there are two bulges, each of them bearing four digging bristles. Non-prominent eye tubercles and numerous mandibular bristles are morphological traits of pit-builders. Most of the behavioural traits are related to pit builders, whereas forward movement, waiting for prey without a pit and frequent changing of ambush location are traits of non-pit builders.
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Bagres/clasificación , Insectos/clasificación , Larva/anatomía & histología , Estructuras Animales/anatomía & histología , Estructuras Animales/crecimiento & desarrollo , Animales , Tamaño Corporal , Bagres/anatomía & histología , Bagres/crecimiento & desarrollo , Femenino , Insectos/anatomía & histología , Insectos/crecimiento & desarrollo , Larva/clasificación , Larva/crecimiento & desarrollo , Masculino , Tamaño de los ÓrganosRESUMEN
Apart from its hematopoietic activity, erythropoietin (EPO) is also known as a tissue-protective cytokine. In the brain, EPO and its receptor are up-regulated in response to insult and exert pro-survival effects. EPO binds to its receptor (EPOR) via high- and low-affinity binding sites (Sites 1 and 2, respectively), inducing conformational changes in the receptor, followed by the activation of downstream signaling cascades. Based on the crystal structure of the EPO:EPOR(2) complex, we designed a peptide, termed Epobis, whose sequence encompassed amino acids from binding Site 1. The present study shows that the Epobis peptide specifically binds to EPOR and induces neurite outgrowth from primary neurons in an EPOR-expression dependent manner. Furthermore, Epobis promoted the survival of hippocampal and cerebellar neuronal cultures after kainate treatment and KCl deprivation, respectively. Thus, we identified a new functional agonist of EPOR with the potential to promote neuroregeneration and neuroprotection.
Asunto(s)
Neuritas/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Péptidos/farmacología , Receptores de Eritropoyetina/agonistas , Receptores de Eritropoyetina/metabolismo , Animales , Western Blotting , Supervivencia Celular/efectos de los fármacos , Eritropoyetina/química , Eritropoyetina/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Modelos Moleculares , Fármacos Neuroprotectores/metabolismo , Péptidos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Ratas , Ratas Wistar , Transducción de Señal/fisiología , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
The fibroblast growth factor receptor (FGFR) plays a vital role in the development of the nervous system regulating a multitude of cellular processes. One of the interaction partners of the FGFR is the neural cell adhesion molecule (NCAM), which is known to play an important role in neuronal development, regeneration and synaptic plasticity. Thus, simultaneous activation of FGFR- and NCAM-mediated signaling pathways may be expected to affect processes underlying neurodegenerative diseases. We here report the identification of a peptide compound, Enreptin, capable of interacting with both FGFR and NCAM. We demonstrate that this dual specificity agonist induces phosphorylation of FGFR and differentiation and survival of primary neurons in vitro, and that these effects are inhibited by abrogation of both NCAM and FGFR signaling pathways. Furthermore, Enreptin crosses the blood-brain barrier after subcutaneous administration, enhances long-term memory in normal mice and ameliorates memory deficit in mice with induced brain inflammation. Moreover, Enreptin reduces cognitive impairment and neuronal death induced by Aß25-35 in a rat model of Alzheimer's disease, and reduces the mortality rate and clinical signs of experimental autoimmune encephalomyelitis in rats. Thus, Enreptin is an attractive candidate for the treatment of neurological diseases.
Asunto(s)
Memoria/efectos de los fármacos , Moléculas de Adhesión de Célula Nerviosa/agonistas , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Animales , Conducta Animal/efectos de los fármacos , Encefalopatías/patología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Neuronas/citología , Ratas , Ratas Wistar , Resonancia por Plasmón de SuperficieRESUMEN
The neural cell adhesion molecule (NCAM) is known to play a role in developmental and structural processes but also in synaptic plasticity and memory of the adult animal. Recently, FGL, a NCAM mimetic peptide that binds to the Fibroblast Growth Factor Receptor 1 (FGFR-1), has been shown to have a beneficial impact on normal memory functioning, as well as to rescue some pathological cognitive impairments. Whether its facilitating impact may be mediated through promoting neuronal plasticity is not known. The present study was therefore designed to test whether FGL modulates the induction and maintenance of synaptic plasticity in the dentate gyrus (DG) in vivo. For this, we first assessed the effect of the FGL peptide on synaptic functions at perforant path-dentate gyrus synapses in the anesthetized rat. FGL, or its control inactive peptide, was injected locally 60 min before applying high-frequency stimulation (HFS) to the medial perforant path. The results suggest that although FGL did not alter basal synaptic transmission, it facilitated both the induction and maintenance of LTP. Interestingly, FGL also modified the heterosynaptic plasticity observed at the neighboring lateral perforant path synapses. The second series of experiments, using FGL intracerebroventricular infusion in the awake animal, confirmed its facilitating effect on LTP for up to 24 h. Our data also suggest that FGL could alter neurogenesis associated with LTP. In sum, these results show for the first time that enhancing NCAM functions by mimicking its heterophilic interaction with FGFR facilitates hippocampal synaptic plasticity in the awake, freely moving animal.
Asunto(s)
Giro Dentado/fisiología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Plasticidad Neuronal/fisiología , Animales , Giro Dentado/efectos de los fármacos , Moléculas de Adhesión de Célula Nerviosa/farmacología , Plasticidad Neuronal/efectos de los fármacos , Ratas , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
The ability of locusts to detect looming stimuli and avoid collisions or predators depends on a neuronal circuit in the locust's optic lobe. Although comprehensively studied for over three decades, there are still major questions about the computational steps of this circuit. We used fourth instar larvae of Locusta migratoria to describe the connection between the lobula giant movement detector 1 (LGMD1) neuron in the lobula complex and the upstream neuropil, the medulla. Serial block-face scanning electron microscopy (SBEM) was used to characterize the morphology of the connecting neurons termed trans-medullary afferent (TmA) neurons and their synaptic connectivity. This enabled us to trace neurons over several hundred micrometers between the medulla and the lobula complex while identifying their synapses. We traced two different TmA neurons, each from a different individual, from their synapses with the LGMD in the lobula complex up into the medulla and describe their synaptic relationships. There is not a simple downstream transmission of the signal from a lamina neuron onto these TmA neurons; there is also a feedback loop in place with TmA neurons making outputs as well as receiving inputs. More than one type of neuron shapes the signal of the TmA neurons in the medulla. We found both columnar and trans-columnar neurons connected with the traced TmA neurons in the medulla. These findings indicate that there are computational steps in the medulla that have not been included in models of the neuronal pathway for looming detection.
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Saltamontes/fisiología , Bulbo Raquídeo/fisiología , Microscopía Electrónica de Rastreo , Neuronas Aferentes/fisiología , Neuronas/fisiología , Vías Visuales/fisiología , Animales , Retroalimentación , Larva , Percepción de Movimiento/fisiología , Lóbulo Óptico de Animales no MamíferosRESUMEN
Neuroplastin-65 (Np65) is a brain-specific cell adhesion molecule belonging to the immunoglobulin superfamily. Homophilic trans-interaction of Np65 mediates adhesion between cells and modulates synaptic plasticity. This interaction solely occurs through the first immunoglobulin (Ig) module of Np65, but the exact binding mechanism has not yet been elucidated. In this study, we identify the homophilic binding motif of Np65 and show that a synthetic peptide modeled after this motif, termed enplastin, binds to Np65. We demonstrate that both Np65- and enplastin-induced intracellular signaling depends on fibroblast growth factor receptor, p38 mitogen-activated protein kinase, Ca(2+) /calmodulin-dependent protein kinase, and cytoplasmic Ca(2+) concentration. In addition, we show that interference with Np65 homophilic binding by enplastin has an inhibitory effect on Np65-mediated neurite outgrowth in vitro and on the initial phase of spatial learning in rats.
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Glicoproteínas de Membrana/fisiología , Plasticidad Neuronal , Neuronas/fisiología , Péptidos/farmacología , Animales , Sitios de Unión , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Cerebelo/citología , Cerebelo/embriología , Cerebelo/metabolismo , Activación Enzimática , Hipocampo/citología , Hipocampo/embriología , Hipocampo/metabolismo , Aprendizaje por Laberinto/efectos de los fármacos , Glicoproteínas de Membrana/genética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Imitación Molecular , Neuritas/efectos de los fármacos , Neuritas/fisiología , Plasticidad Neuronal/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Péptidos/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Neuroplastin (Np) is a glycoprotein belonging to the immunoglobulin superfamily of cell adhesion molecules (CAMs) and existing in two isoforms, Np55 and Np65, named according to their molecular weights. The extracellular part of Np65 contains three immunoglobulin (Ig)-like modules (Ig1, Ig2, and Ig3), whereas Np55 lacks the Ig1 module. Of these two isoforms, only Np65 is involved in homophilic interactions resulting in cell adhesion, whereas the role of Np55 is poorly understood. The present study reports for the first time the crystal structure of the ectodomain of Np55 at 1.95-A resolution and demonstrates that Np55 binds to and activates the fibroblast growth factor receptor 1 (FGFR1). Furthermore, we identify a sequence motif in the Ig2 module of Np55 interacting with FGFR1 and show that a synthetic peptide encompassing this motif, termed narpin, binds to and activates FGFR1. We show that both Np55 and the narpin peptide induce neurite outgrowth through FGFR1 activation and that Np55 increases synaptic calcium concentration in an FGFR1-dependent manner. Moreover, we demonstrate that narpin has an antidepressive-like effect in rats subjected to the forced swim test, suggesting that Np55-induced signaling may be involved in synaptic plasticity in vivo. Owczarek, S., Kiryushko, D., Larsen, M. H., Kastrup, J. S., Gajhede, M., Sandi, C., Berezin, V., Bock, E., Soroka, V. Neuroplastin-55 binds to and signals through the fibroblast growth factor receptor.
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Calcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Péptidos/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/fisiología , Sinapsis/metabolismo , Secuencias de Aminoácidos , Animales , Antidepresivos/química , Antidepresivos/metabolismo , Antidepresivos/farmacología , Línea Celular , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/farmacología , Neuritas/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Péptidos/química , Péptidos/farmacología , Condicionamiento Físico Animal , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , NataciónRESUMEN
Erythropoietin, a member of the type 1 cytokine superfamily, controls proliferation and differentiation of erythroid progenitor cells through binding to and dimerization of the erythropoietin receptor. Both erythropoietin and its receptor are also expressed in the central nervous system, where they are involved in tissue protection. However, the use of erythropoietin as a neuroprotective agent may be hampered by its erythropoietic activity. Therefore, developing non-haematopoietic erythropoietin mimetics is important. Based on the crystal structure of the complex of erythropoietin and its receptor, we designed a peptide, termed Epotris, corresponding to the C α-helix region (amino-acid residues 92-111) of human erythropoietin. The peptide specifically bound to the erythropoietin receptor and promoted neurite outgrowth and survival of primary neurons with the same efficiency as erythropoietin, but with 10(3)-fold lower potency. Knockdown of the erythropoietin receptor or interference with its downstream signalling inhibited the Epotris-induced neuritogenic and pro-survival effect. Similarly to erythropoietin, Epotris penetrated the blood-brain barrier. Moreover, treatment with the peptide attenuated seizures, decreased mortality and reduced neurodegeneration in an in vivo model of kainic acid-induced neurotoxicity. In contrast to erythropoietin, Epotris did not stimulate erythropoiesis upon chronic administration. Thus, Epotris is a novel neuroprotective non-haematopoietic erythropoietin mimetic that may offer new opportunities for the treatment of neurological disorders.
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Eritropoyetina/farmacología , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/farmacología , Receptores de Eritropoyetina/agonistas , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Células Cultivadas , Eritropoyesis/efectos de los fármacos , Eritropoyesis/fisiología , Eritropoyetina/química , Eritropoyetina/metabolismo , Eritropoyetina/farmacocinética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neuritas/efectos de los fármacos , Neuritas/fisiología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/mortalidad , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacocinética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacocinética , Ratas , Ratas Wistar , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/mortalidadRESUMEN
The formation of appropriate neuronal circuits is an essential part of nervous system development and relies heavily on the outgrowth of axons and dendrites and their guidance to their respective targets. This process is governed by a large array of molecules, including glial cell line-derived neurotrophic factor (GDNF) and the neural cell adhesion molecule (NCAM), the interaction of which induce neurite outgrowth. In the present study the requirements for NCAM-mediated GDNF-induced neurite outgrowth were investigated in cultures of hippocampal neurons, which do not express Ret. We demonstrate that NCAM-mediated GDNF-induced signaling leading to neurite outgrowth is more complex than previously reported. It not only involves NCAM-140 and the Src family kinase Fyn but also uses NCAM-180 and the fibroblast growth factor receptor. We find that induction of neurite outgrowth by GDNF via NCAM or by trans-homophilic NCAM interactions are not mutually exclusive. However, whereas NCAM-induced neurite outgrowth primarily is mediated by NCAM-180, we demonstrate that GDNF-induced neurite outgrowth involves both NCAM-140 and NCAM-180. We also find that GDNF-induced neurite outgrowth via NCAM differs from NCAM-induced neurite outgrowth by being independent of NCAM polysialylation. Additionally, we investigated the structural basis for GDNF-NCAM interactions and find that NCAM Ig3 is necessary for GDNF binding. Furthermore, we identify within the heel region of GDNF a binding site for NCAM and demonstrate that a peptide encompassing this sequence mimics the effects of GDNF with regard to NCAM binding, activation of intracellular signaling, and induction of neurite outgrowth.
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Factor Neurotrófico Derivado de la Línea Celular Glial/fisiología , Moléculas de Adhesión de Célula Nerviosa/fisiología , Neuritas/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Línea Celular , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Ratas , Ratas WistarRESUMEN
Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic biologic actions by binding, dimerizing, and activating FGF receptors (FGFRs). The present study reports the physiologic impact of various FGF2-FGFR1 contact sites employing three different synthetic peptides, termed canofins, designed based on structural analysis of the interactions between FGF2 and FGFR1. Canofins mimic the cognate ligand interaction with the receptor and preserve the neuritogenic and neuroprotective properties of FGF2. Canofins were shown by surface plasmon resonance analysis to bind to FGFR1 and promote receptor activation. However, FGF2-induced receptor phosphorylation was inhibited by canofins, indicating that canofins are partial FGFR agonists. Furthermore, canofins were demonstrated to induce neuronal differentiation determined by neurite outgrowth from cerebellar granule neurons, and this effect was dependent on FGFR activation. Additionally, canofins acted as neuroprotectants, promoting survival of cerebellar granule neurons induced to undergo apoptosis. Our results suggest that canofins mirror the effect of specific interaction sites in FGF2 for FGFR. Thus, canofins are valuable pharmacological tools to study the functional roles of specific molecular interactions of FGF2 with FGFR.
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Dendrímeros/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Neuronas/efectos de los fármacos , Oligopéptidos/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Dendrímeros/química , Agonismo Parcial de Drogas , Factor 2 de Crecimiento de Fibroblastos/química , Ligandos , Modelos Moleculares , Neuritas/efectos de los fármacos , Neuritas/fisiología , Neuronas/citología , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Oligopéptidos/química , Fosforilación , Ratas , Ratas Wistar , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/agonistas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Resonancia por Plasmón de SuperficieRESUMEN
Activation of fibroblast growth factor (FGF) receptors (FGFRs) both by FGFs and by the neural cell adhesion molecule (NCAM) is crucial in the development and function of the nervous system. We found that FGFR substrate 2alpha (FRS2alpha), Src homologous and collagen A (ShcA), and phospholipase-Cgamma (PLCgamma) were all required for neurite outgrowth from cerebellar granule neurons (CGNs) induced by FGF1 and FGL (an NCAM-derived peptide agonist of FGFR1). Like FGF1, FGL induced tyrosine phosphorylation of FGFR1, FRS2alpha, ShcA, and PLCgamma in a time- and dose-dependent manner. However, the activation of FRS2alpha by FGL was significantly lower than the activation by FGF1, indicating a differential signaling profile induced by NCAM compared with the cognate growth factor.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cerebelo/fisiología , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/fisiología , Animales , Aumento de la Célula , Células Cultivadas , Modelos Neurológicos , Neuritas/fisiología , Fosfolipasa C gamma/metabolismo , Fosforilación , Ratas , Ratas Wistar , Receptores de Factores de Crecimiento de Fibroblastos/agonistas , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Factores de Tiempo , Tirosina/metabolismoRESUMEN
The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and synaptic plasticity. The crystal structure of a fragment of NCAM comprising the three N-terminal immunoglobulin (Ig)-like modules indicates that the first and second Ig modules bind to each other, thereby presumably mediating dimerization of NCAM molecules expressed on the same cell surface (cis-interactions), whereas the third Ig module, through interactions with the first or second Ig module, mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans-interactions). We have designed a new potent peptide ligand of NCAM, termed plannexin, based on a discontinuous sequence in the second NCAM Ig module that represents a homophilic binding site for an opposing third Ig module. The peptide was found by surface plasmon resonance analysis to bind the third NCAM Ig module. It promoted survival of cultured cerebellar granule neurons (CGNs) and also induced neurite extension in cultures of dopaminergic neurons and CGNs; the latter effect was shown to be dependent on NCAM expression, indicating that plannexin mimics the neuritogenic effect of homophilic NCAM binding.