RESUMEN
The retina is the sensory tissue responsible for the first stages of visual processing, with a conserved anatomy and functional architecture among vertebrates. To date, retinal eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, and others, affect nearly 170 million people worldwide, resulting in vision loss and blindness. To tackle retinal disorders, the developing retina has been explored as a versatile model to study intercellular signaling, as it presents a broad neurochemical repertoire that has been approached in the last decades in terms of signaling and diseases. Retina, dissociated and arranged as typical cultures, as mixed or neuron- and glia-enriched, and/or organized as neurospheres and/or as organoids, are valuable to understand both neuronal and glial compartments, which have contributed to revealing roles and mechanisms between transmitter systems as well as antioxidants, trophic factors, and extracellular matrix proteins. Overall, contributions in understanding neurogenesis, tissue development, differentiation, connectivity, plasticity, and cell death are widely described. A complete access to the genome of several vertebrates, as well as the recent transcriptome at the single cell level at different stages of development, also anticipates future advances in providing cues to target blinding diseases or retinal dysfunctions.
Asunto(s)
Enfermedades de la Retina , Animales , Humanos , Ceguera , Estado de Salud , Neuroglía , Neuronas , RetinaRESUMEN
Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, therapeutic strategies are needed to protect and replace these projection neurons. One innovative approach is to promote de novo genesis of RGCs via manipulation of endogenous cell sources. Here, we demonstrate that the pluripotency regulator gene Krüppel-like factor 4 (Klf4) is sufficient to change the potency of lineage-restricted retinal progenitor cells to generate RGCs in vivo Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2 In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30â days after in vivo generation. We identified Klf4 as a promising candidate for reprogramming retinal cells and regenerating RGCs in the retina.This article has an associated 'The people behind the papers' interview.
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Factores de Transcripción de Tipo Kruppel/fisiología , Neurogénesis , Células Ganglionares de la Retina/fisiología , Animales , Ciclo Celular , Femenino , Proteínas de Homeodominio/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regeneración Nerviosa , Células-Madre Neurales/fisiología , Ratas , Factor de Transcripción Brn-3A/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Pez Cebra , Proteínas de Pez Cebra/fisiologíaRESUMEN
Schizophrenia etiology is unknown, nevertheless imbalances occurring in an acute psychotic episode are important to its development, such as alterations in cellular energetic state, REDOX homeostasis and intracellular Ca2+ management, all of which are controlled primarily by mitochondria. However, mitochondrial function was always evaluated singularly, in the presence of specific respiratory substrates, without considering the plurality of the electron transport system. In this study, mitochondrial function was analyzed under conditions of isolated or multiple respiratory substrates using brain mitochondria isolated from MK-801-exposed mice. Results showed a high H2O2 production in the presence of pyruvate/malate, with no change in oxygen consumption. In the condition of multiple substrates, however, this effect is lost. The analysis of Ca2+ retention capacity revealed a significant change in the uptake kinetics of this ion by mitochondria in MK-801-exposed animals. Futhermore, when mitochondria were exposed to calcium, a total loss of oxidative phosphorylation and an impressive increase in H2O2 production were observed in the condition of multiple substrates. There was no alteration in the activity of the antioxidant enzymes analyzed. The data demonstrate for the first time, in an animal model of psychosis, two important aspects (1) mitochondria may compensate deficiencies in a single mitochondrial complex when they oxidize several substrates simultaneously, (2) Ca2+ handling is compromised in MK-801-exposed mice, resulting in a loss of phosphorylative capacity and an increase in H2O2 production. These data favor the hypothesis that disruption of key physiological roles of mitochondria may be a trigger in acute psychosis and, consequently, schizophrenia.
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Encéfalo/patología , Calcio/efectos adversos , Mitocondrias/patología , Trastornos Psicóticos/complicaciones , Enfermedad Aguda , Animales , Humanos , Masculino , RatonesRESUMEN
Pituitary Adenylyl Cyclase-Activating Polypeptide (PACAP) is a neuroactive peptide present in the avian retina where it activates adenylyl cyclase (AC) since early in development via PACAP receptors. The synthesis of cAMP in response to PACAP is observed since embryonic day 8/9 (E8/9). After E12, signaling via PACAP receptors desensitizes, reaching very low levels in the mature tissue. We show here that chronic administration of PACAP in vitro desensitizes PACAP-induced cAMP accumulation, while the administration of the PACAP antagonist (PACAP 6-38) re-sensitizes PACAP receptor/cyclase system in vitro and in vivo. Moreover, a twofold increase in the number of tyrosine hydroxylase positive (THâº) cells is observed after in vivo injection of PACAP6-38. NURR1, a transcription factor associated with the differentiation of dopaminergic cells in the CNS, is present in the chick retina in all developmental stages studied. The presence of NURR1 positive cells in the mature tissue far exceeds the number of TH⺠cells, suggesting that these NURR1-positive cells might have the potential to express the dopaminergic phenotype. Our data show that if PACAP signaling is increased in mature retinas, plastic changes in dopaminergic phenotype can be achieved.
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Plasticidad Neuronal/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Retina/metabolismo , Animales , Western Blotting , Pollos , AMP Cíclico , Dopamina , Inmunohistoquímica , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Microglia are the most prominent immune resident cell population in the central nervous system (CNS). In the healthy CNS, microglia survey their surrounding microenvironment, through recurrent extension and retraction of filopodia-like membrane protrusions, without evident cell body displacement. Microglia undergo dramatic transcriptomic and shape changes upon brain insults or neurodegenerative disease states and adopt a classical immune effector function (producing an extensive array of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species) to re-establish tissue homeostasis. While the biophysical principles underlying microglia morphological changes remain elusive, several recent studies have highlighted the pivotal role of the actin and non-muscle myosin II filamentous cytoskeleton in this process. In this work, we discuss how subcellular topological patterning of the actin and myosin cytoskeleton can control microglial cell shape dynamics and how it can potentially feedback on their functional specialization, which is of great importance to understanding the mechanisms of microglial action in homeostatic conditions and CNS disease states.
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Microglía , Enfermedades Neurodegenerativas , Actinas/metabolismo , Encéfalo/metabolismo , Humanos , Microglía/metabolismo , Neuronas/metabolismoRESUMEN
Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism that are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In the present study, we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D1R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H2O2 and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from induced-pluripotent stem cells (iPSCs) of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast with NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia.
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Dopamina/farmacología , Hexoquinasa/metabolismo , Mitocondrias/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Receptores de Dopamina D1/agonistas , Esquizofrenia/enzimología , Calcio/metabolismo , Estudios de Casos y Controles , Línea Celular , Humanos , Mitocondrias/enzimología , Células-Madre Neurales/enzimología , Receptores de Dopamina D1/metabolismo , Transducción de SeñalRESUMEN
During retinal development, cell proliferation and exit from the cell cycle must be precisely regulated to ensure the generation of the appropriate numbers and proportions of the various retinal cell types. Previously, we showed that pituitary adenylyl cyclase-activating polypeptide (PACAP) exerts a neuroprotective effect in the developing retina of rats, through the cAMP-cAMP-dependent protein kinase (protein kinase A) (PKA) pathway. Here, we show that PACAP also regulates the proliferation of retinal progenitor cells. PACAP, PACAP-specific receptor (PAC1), and the receptors activated by both PACAP and vasoactive intestinal peptide (VIP), VPAC1 and VPAC2, are expressed during embryonic and postnatal development of the rat retina. Treatment of retinal explants with PACAP38 reduced the incorporation of [(3)H]thymidine as well as the number of 5-bromo-2'-deoxyuridine-positive and cyclin D1-positive cells. Pharmacological experiments indicated that PACAP triggers this antiproliferative effect through the activation of both PAC1 and VPACs, and the cAMP-PKA pathway. In addition, PACAP receptor activation decreased both cyclin D1 mRNA and protein content. Altogether, the data support the hypothesis that PACAP is a cell-extrinsic regulator with multiple roles during retinal development, including the regulation of proliferation in a subpopulation of retinal progenitor cells.
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Proliferación Celular , Ciclina D1/metabolismo , Regulación hacia Abajo , Neurogénesis/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Retina/metabolismo , Neuronas Retinianas/metabolismo , Análisis de Varianza , Animales , Western Blotting , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclina D1/genética , Inmunohistoquímica , Microscopía Confocal , Fosforilación , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Receptores de Péptido Intestinal Vasoactivo/genética , Receptores de Péptido Intestinal Vasoactivo/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre , Técnicas de Cultivo de Tejidos , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Macrophage migration inhibitory factor (MIF) participates in the pathogenesis of inflammatory diseases, including asthma, in which it enhances airway hypersensitivity and tissue eosinophilia. Herein, we investigated the role of MIF in eosinophilopoiesis and tissue eosinophilia using Schistosoma mansoni infection. MIF-deficient (Mif(-/-)) mice had similar numbers of adult worms, eggs, and granulomas compared to wild-type mice, but the size of granulomas was strikingly reduced due to smaller numbers of eosinophils. MIF did not affect the acquired response to infection, as Mif(-/-) mice produced normal amounts of Th2 cytokines and IgE. Nevertheless, recombinant MIF (rMIF) behaved as a chemoattractant for eosinophils, what could partially explain the reduced eosinophilia in infected Mif(-/-) mice. Moreover, the percentage of eosinophils was reduced in bone marrows of Mif(-/-) mice chronically infected with S. mansoni compared to wild type. Mif(-/-) had impaired eosinophilopoiesis in response to interleukin (IL)-5 and addition of rMIF to bone marrow cultures from IL-5 transgenic mice enhanced the generation of eosinophils. In the absence of MIF, eosinophil precursors were unable to survive the IL-5-supplemented cell culture, and were ingested by macrophages. Treatment with pancaspase inhibitor z-VAD or rMIF promoted the survival of eosinophil progenitors. Together, these results indicate that MIF participates in IL-5-driven maturation of eosinophils and in tissue eosinophilia associated with S. mansoni infection.
Asunto(s)
Eosinofilia/inmunología , Eosinófilos/inmunología , Interleucina-5/fisiología , Factores Inhibidores de la Migración de Macrófagos/fisiología , Esquistosomiasis mansoni/patología , Animales , Eosinofilia/etiología , Eosinofilia/patología , Eosinófilos/patología , Granuloma/etiología , Granuloma/inmunología , Granuloma/patología , Inflamación/patología , Interleucina-5/inmunología , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Recombinantes/inmunología , Esquistosomiasis mansoni/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Retinal development follows a conserved neurogenic program in vertebrates to orchestrate the generation of specific cell types from multipotent progenitors in sequential but overlapping waves. In this program, retinal ganglion cells (RGCs) are the first cell type generated. RGCs are the final output neurons of the retina and are essential for vision and circadian rhythm. Key molecular steps have been defined in multiple vertebrate species to regulate competence, specification, and terminal differentiation of this cell type. This involves neuronal-specific transcription factor networks, regulators of chromatin dynamics and miRNAs. In mammals, RGCs and their optic nerve axons undergo neurodegeneration and loss in glaucoma and other optic neuropathies, resulting in irreversible vision loss. The incapacity of RGCs and axons to regenerate reinforces the need for the design of efficient RGC replacement strategies. Here we describe the essential molecular pathways for the differentiation of RGCs in vertebrates, as well as experimental manipulations that extend the competence window for generation of this early cell type from late progenitors. We discuss recent advances in regeneration of retinal neurons in vivo in both mouse and zebrafish and discuss possible strategies and barriers to achieving RGC regeneration as a therapeutic approach for vision restoration in blinding diseases such as glaucoma.
RESUMEN
Degenerative retinopathies are the leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Müller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the rodent adult injured retina but contribute weakly to tissue repair when compared to zebrafish retina. However, postnatal and adult mouse MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram postnatal mouse MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1 and both TFs induce the generation of functionally active iNs. Treatment of MGC cultures with EGF and FGF2 prior to Neurog2 or Ascl1 expression enhances reprogramming efficiencies, what can be at least partially explained by an increase in the frequency of MGCs expressing sex determining region Y (SRY)-box 2 (SOX2). Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. Moreover, in vivo electroporation of Neurog2 in late progenitors from the neonatal rat retina, which are transcriptionally similar to MGCs, also induced a shift in the generation of retinal cell subtypes, favoring neuronal differentiation at the expense of MGCs and resuming the generation of RGCs. Altogether, our data indicate that NEUROG2 induces lineage conversion of postnatal rodent MGCs into RGC-like iNs in vitro and resumes the generation of this neuronal type from late progenitors of the retina in vivo.
RESUMEN
It has long been known that a barrage of signals from neighboring and connecting cells, as well as components of the extracellular matrix, control cell survival. Given the extensive repertoire of retinal neurotransmitters, neuromodulators and neurotrophic factors, and the exhuberant interconnectivity of retinal interneurons, it is likely that various classes of released neuroactive substances may be involved in the control of sensitivity to retinal cell death. The aim of this article is to review evidence that neurotransmitters and neuropeptides control the sensitivity to programmed cell death in the developing retina. Whereas the best understood mechanism of execution of cell death is that of caspase-mediated apoptosis, current evidence shows that not only there are many parallel pathways to apoptotic cell death, but non-apoptotic programs of execution of cell death are also available, and may be triggered either in isolation or combined with apoptosis. The experimental data show that many upstream signaling pathways can modulate cell death, including those dependent on the second messengers cAMP-PKA, calcium and nitric oxide. Evidence for anterograde neurotrophic control is provided by a variety of models of the central nervous system, and the data reviewed here indicate that an early function of certain neurotransmitters, such as glutamate and dopamine, as well as neuropeptides such as pituitary adenylyl cyclase-activating polypeptide and vasoactive intestinal peptide is the trophic support of cell populations in the developing retina. This may have implications both regarding the mechanisms of retinal organogenesis, as well as pathological conditions leading to retinal dystrophies and to dysfunctional cellular behavior.
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Apoptosis/fisiología , Neuropéptidos/fisiología , Neurotransmisores/fisiología , Retina/crecimiento & desarrollo , Animales , Supervivencia Celular , Humanos , Retina/citología , Retina/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Neurite outgrowth is a critical event in neuronal development, formation, and remodeling of synapses, response to injury, and regeneration. We examined the effects of 2,4-dinitrophenol (DNP), a recently described blocker of the aggregation and neurotoxicity of the beta-amyloid peptide, on neurite elongation of central neurons. Morphometric analysis of rat embryo hippocampal and cortical neuronal cultures showed that neurite outgrowth was stimulated by DNP. This effect was accompanied by increases in the neuronal levels of the microtubule-associated protein tau and of cyclic adenosine 3',5' monophosphate (cAMP). DNP also promoted cAMP accumulation, increased tau level, neurite outgrowth, and neuronal differentiation in the mouse neuroblastoma cell line N2A. We show that DNP-induced differentiation requires activation of the extracellular signal-regulated kinase (ERK). The finding that DNP promotes neuritogenesis and neuronal differentiation suggests that, in addition to its anti-amyloidogenic actions, it may be a useful lead compound in the development of novel therapeutic approaches targeting neurite dystrophy and synaptic dysfunction in neurodegenerative pathologies such as Alzheimer's disease.
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2,4-Dinitrofenol/farmacología , Amiloide/química , Neuritas/patología , Neuronas/metabolismo , Péptidos beta-Amiloides/química , Animales , Western Blotting , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Corteza Cerebral/patología , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hipocampo/citología , Hipocampo/embriología , Sistema de Señalización de MAP Quinasas , Ratones , Microscopía Fluorescente , Enfermedades Neurodegenerativas/patología , Oxígeno/metabolismo , Consumo de Oxígeno , Fragmentos de Péptidos/química , Ratas , Especies Reactivas de Oxígeno , Factores de Tiempo , Desacopladores/farmacología , Proteínas tau/químicaRESUMEN
Programmed cell death occurs in the nervous system both in normal development as well as in pathologic conditions, and is a key issue related to both brain repair and neurodegenerative diseases. Modulation of cell death in the nervous system may involve neurotrophic factors and other peptides, neurotransmitters and neuromodulators, that activate various signal transduction pathways, which in turn interact with the cell death execution machinery. Here we discuss the role of the second messenger cyclic adenosine 3'5'-monophosphate (cAMP) in cell death, and summarize current evidence that cAMP is a nodal point of neuroprotective signaling pathways.
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Muerte Celular/fisiología , AMP Cíclico/metabolismo , Fármacos Neuroprotectores/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , HumanosRESUMEN
We showed previously that the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) negatively regulates proliferation of postnatal rat retinal progenitor cells through the downregulation of cyclin D1 in a cAMP/protein kinase A dependent manner. In the present study, we describe by microarray analysis several putative PACAP targets regulated by different transcription factor families. One of these families is the Sp/Klf family of transcriptional factors capable of regulating cyclin D1, and among members, we demonstrate by immunocytochemistry that KLF4 is expressed throughout rat retinal development by retinal progenitor cells and in most differentiated cell types. Using retinal explants preparations, PACAP treatment can transiently increase Klf4 mRNA levels; from electrophoretic mobility shift assays, PACAP is also able to increase the nuclear KLF4 content. From these results, we suggest that KLF4 may be involved in the anti-proliferative effects of PACAP as one mechanism regulating progenitor cell transition from proliferation to differentiation throughout retinal development.
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Proliferación Celular , Pleiotropía Genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Retina/metabolismo , Animales , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/fisiología , Ratas , Ratas Sprague-Dawley , Retina/citología , Retina/crecimiento & desarrolloRESUMEN
BACKGROUND: Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. METHODOLOGY/PRINCIPAL FINDINGS: We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (ß-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. CONCLUSION: Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.
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Western Blotting/métodos , Estándares de Referencia , Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Perfilación de la Expresión Génica/métodos , Técnicas In Vitro , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , ARN Mensajero/genética , Ratas , Programas InformáticosRESUMEN
Hop/STI1 is a co-chaperone adaptor protein for Hsp70/Hsp90 complexes. Hop/STI1 is found extracellularly and modulates cell death and differentiation through interaction with the prion protein (PrP(C)). Here, we investigated the expression of hop/STI1 and its role upon cell proliferation and cell death in the developing retina. Hop/STI1 is more expressed in developing rat retina than in the mature tissue. Hop/STI1 blocks retinal cell death in the neuroblastic layer (NBL) in a PrP(C) dependent manner, but failed to protect ganglion cells against axotomy-induced cell death. An antibody raised against hop/STI1 (alpha-STI1) blocked both ganglion cell and NBL cell death independent of PrP(C). cAMP/PKA, ERK, PI3K and PKC signaling pathways were not involved in these effects. Hop/STI1 treatment reduced proliferation, while alpha-STI1 increased proliferation in the developing retina, both independent of PrP(C). We conclude that hop/STI1 can modulate both proliferation and cell death in the developing retina independent of PrP(C).
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Proteínas de Choque Térmico/metabolismo , Retina/citología , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Muerte Celular , Proliferación Celular/efectos de los fármacos , Citoprotección/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Ratones , Proteínas PrPC/metabolismo , Ratas , Retina/efectos de los fármacos , Retina/embriología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacosRESUMEN
Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.
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Glicoproteínas/química , Glicoproteínas/fisiología , Neuraminidasa/química , Neuraminidasa/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/fisiología , Trypanosoma cruzi/patogenicidad , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Glicoproteínas/genética , Humanos , Queratina-18/química , Queratina-18/metabolismo , Sistema de Señalización de MAP Quinasas , Neuraminidasa/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , VirulenciaRESUMEN
Glutamate is a classical excitotoxin of the central nervous system (CNS), but extensive work demonstrates neuroprotective roles of this neurotransmitter in developing CNS. Mechanisms of glutamate-mediated neuroprotection are still under scrutiny. In this study, we investigated mediators of glutamate-induced neuroprotection, and tested whether this neurotransmitter controls programmed cell death in the developing retina. The protective effect of N-methyl-d-aspartate (NMDA) upon differentiating cells of retinal explants was completely blocked by a neutralizing antibody to brain-derived neurotrophic factor (BDNF), but not by an antibody to neurotrophin-4 (NT-4). Consistently, chronic activation of NMDA receptor increased the expression of BDNF and trkB mRNA, as well as BDNF protein content, but did not change the content of NT-4 mRNA in retinal tissue. Furthermore, we showed that in vivo inactivation of NMDA receptor by intraperitoneal injections of MK-801 increased natural cell death of specific cell populations of the post-natal retina. Our results show that chronic activation of NMDA receptors in vitro induces a BDNF-dependent neuroprotective state in differentiating retinal cells, and that NMDA receptor activation controls programmed cell death of developing retinal neurons in vivo.
Asunto(s)
Envejecimiento/fisiología , Animales Recién Nacidos/fisiología , Apoptosis/fisiología , Factor Neurotrófico Derivado del Encéfalo/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Retina/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Apoptosis/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/genética , Maleato de Dizocilpina/farmacología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , N-Metilaspartato/farmacología , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/fisiología , Fármacos Neuroprotectores/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Receptor trkB/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Retina/crecimiento & desarrolloRESUMEN
Multiple neuroactive substances are secreted by neurons and/or glial cells and modulate the sensitivity to cell death. In the developing retina, it has been shown that increased intracellular levels of cAMP protect cells from degeneration. We tested the hypothesis that the neuroactive peptide pituitary adenylyl cyclase-activating polypeptide (PACAP) has neuroprotective effects upon the developing rat retina. PACAP38 prevented anisomycin-induced cell death in the neuroblastic layer (NBL) of retinal explants, and complete inhibition of induced cell death was obtained with 1 nm. A similar protective effect was observed with PACAP27 and with the specific PAC1 receptor agonist maxadilan but not with glucagon. Photoreceptor cell death induced by thapsigargin was also prevented by PACAP38. The neuroprotective effect of PACAP38 upon the NBL could be reverted by the competitive PACAP receptor antagonist PACAP6-38 and by the specific PAC1 receptor antagonist Maxd.4. Molecular and immunohistochemical analysis demonstrated PAC1 receptors, and treatment with PACAP38 induced phospho-cAMP-response element-binding protein immunoreactivity in the anisomycin-sensitive undifferentiated postmitotic cells within the NBL. PACAP38 produced an increase in cAMP but not inositol triphosphate, and treatment with the cAMP-dependent protein kinase inhibitor R(p)-cAMPS blocked the protective effect of PACAP38. The results indicate that activation of PAC1 receptors by PACAP38 modulates cell death in the developing retina through the intracellular cAMP/cAMP-dependent protein kinase pathway.
Asunto(s)
Muerte Celular/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuropéptidos/farmacología , Retina/enzimología , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Activación Enzimática , Fármacos Neuroprotectores/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Retina/citología , Retina/efectos de los fármacos , Transcripción GenéticaRESUMEN
Este estudo tem como objetivo avaliar o efeito das técnicas Terapia Expiratória Manual Passiva (TEMP) e de Aumento de Fluxo Expiratório Lento (AFEL) nos fluxos expiratórios. Seis lactentes submetidos às duas técnicas fisioterápicas. Os dados dos fluxos expiratórios foram coletados através de pneumotacógrafo conectado a uma máscara e os dados registrados em um computador. TEMP E AFEL produzem um aumento de fluxos expiratórios em relação aos fluxos gerados durante respiração normal. Não houve significativas entre as duas técnicas quanto incremento de fluxo expiratório. As técnicas TEMP E AFEL não apresentaram magnitude significativa em seus picos de fluxos expiratórios.