RESUMEN
Photoreceptor neurons (R cells) in the Drosophila eye define a map of visual space by connecting to targets in distinct layers of the optic lobe, with R1-6 cells connecting to the lamina (the first optic ganglion) and R7 and R8 cells connecting to the medulla (the second optic ganglion). Here, we show that Wengen (Wgn) directly binds Moesin (Moe) through a cytosolic membrane proximal domain and this interaction is important for mediating two distinct aspects of axonal targeting. First, we show that loss of wgn or moe function disrupts cell autonomous R8 axon targeting. Second, we report that wgn or moe mutants show defects in R2-R5 targeting that result from disruption of non-cell autonomous effects, which are secondary to the cell autonomous R8 phenotype. Thus, these studies reveal that the Wgn-Moe signaling cascade plays a key role in photoreceptor target field innervations through cell autonomous and non-cell autonomous mechanisms.
Asunto(s)
Axones/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Proteínas de Microfilamentos/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Secuencia de Aminoácidos , Animales , Linaje de la Célula , Proteínas de Drosophila/química , Drosophila melanogaster/citología , Técnicas de Silenciamiento del Gen , Conos de Crecimiento/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Neuroglía/citología , Neuroglía/metabolismo , Células Fotorreceptoras de Invertebrados/citología , Unión Proteica , Receptores del Factor de Necrosis Tumoral/químicaRESUMEN
OBJECTIVE: Fas (CD95), commonly categorised as a death receptor due to its well-defined role in apoptosis, can paradoxically also promote neuroprotection. We have previously found that defects in Fas signalling render mice highly susceptible to neural degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD). Decreased activity of the ubiquitin proteasome system and accumulation of protein aggregates are implicated in PD pathogenesis. Here, we investigate the relationship between Fas and ubiquitin proteasomal activity in neuronal cells. METHODS: We performed proteasome assays in neuroblastoma cells and in midbrain cultures of wild-type and Fas-deficient mice. RESULTS: Neuroblastoma cells upregulated proteasomal activity in response to an activating Fas antibody in vitro. Furthermore, neural tissue from Fas-deficient mice showed decreased proteasomal activity compared with the tissue from wild-type mice when exposed to a PD-inducing toxin in vivo. CONCLUSION: These findings suggest that mechanisms for Fas-mediated neuroprotection may include Fas-induced upregulation of proteasomal activity, and consequently less accumulation of toxic protein aggregates.
RESUMEN
Fas ligand (FasL) is best known for its role in apoptosis. Membrane-bound FasL can signal in FasL-bearing cells, a process known as reverse signalling. The biological and functional consequences of FasL reverse signalling in Schwann cells were studied. FasL engagement induced the secretion of soluble mediator(s) that stimulated neurite growth in PC12 cells, NGF secretion, and NGF mRNA levels. ERK1/2 and Src phosphorylation was rapidly increased and inhibition of their activation affected NGF synthesis and release. FasL can therefore act as a signal-transducing molecule in Schwann cells, leading to the secretion of NGF, and may contribute to peripheral nerve regeneration.
Asunto(s)
Proteína Ligando Fas/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Células de Schwann/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Humanos , Ratones , Ratones Endogámicos C57BL , Neuritas/metabolismo , Células PC12 , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A prolonged activation of the immune system is one of the main causes of hyperproliferation of lymphocytes leading to defects in immune tolerance and autoimmune diseases. Fas ligand (FasL), a member of the TNF superfamily, plays a crucial role in controlling this excessive lymphoproliferation by inducing apoptosis in T cells leading to their rapid elimination. Here, we establish that posttranscriptional regulation is part of the molecular mechanisms that modulate FasL expression, and we show that in activated T cells FasL mRNA is stable. Our sequence analysis indicates that the FasL 3'-untranslated region (UTR) contains two AU-rich elements (AREs) that are similar in sequence and structure to those present in the 3'-UTR of TNFα mRNA. Through these AREs, the FasL mRNA forms a complex with the RNA-binding protein HuR both in vitro and ex vivo. Knocking down HuR in HEK 293 cells prevented the phorbol 12-myristate 13-acetate-induced expression of a GFP reporter construct fused to the FasL 3'-UTR. Collectively, our data demonstrate that the posttranscriptional regulation of FasL mRNA by HuR represents a novel mechanism that could play a key role in the maintenance and proper functioning of the immune system.
Asunto(s)
Regiones no Traducidas 3' , Antígenos de Superficie/metabolismo , Proteína Ligando Fas/biosíntesis , Regulación de la Expresión Génica , Estabilidad del ARN , Proteínas de Unión al ARN/metabolismo , Linfocitos T/metabolismo , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Apoptosis/genética , Apoptosis/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Carcinógenos/farmacología , Proteínas ELAV , Proteína 1 Similar a ELAV , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Microglia are resident cells of the CNS that belong to the myeloid cell lineage. In experimental models of neuroinflammation, they have limited capacity to function as APCs when compared with dendritic cells (DCs). Human peripheral blood monocytes have the plasticity to differentiate into mature DCs when exposed to GM-CSF and IL-4 followed by LPS. In this study we addressed the potential of human microglia to acquire phenotypic and functional properties of mature DCs under similar inducing conditions. Treated adult and fetal microglia became CD14(low) and acquired limited expression of CD209 (DC-SIGN); they remained CD1a(-) and CD83(-), and decreased MHCII expression, suggesting that they had not achieved a complete DC phenotype. The monocyte-derived DCs efficiently promoted CD4 T cell proliferation in an allogeneic MLR, whereas differentiated adult microglia had a decreased ability to stimulate CD4 T cell proliferation compared with their untreated counterparts. Differentiated fetal microglia did support CD4 T cell proliferation, whereas untreated cells could not. Fetal and adult microglia produced significant amounts of IL-10 following differentiation but no detectable IL-12 p70, in contrast to differentiated monocytes that produced IL-12 p70. Our data indicate that neither adult nor fetal microglia acquired the full characteristic phenotype of mature stimulatory DCs when treated with DC-inducing cytokines in vitro. Moreover, such treatment, especially of adult microglia, induces functional responses that could promote an antiinflammatory environment in the CNS.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Mediadores de Inflamación/metabolismo , Microglía/inmunología , Microglía/metabolismo , Transducción de Señal/inmunología , Adulto , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/patología , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Citocinas/metabolismo , Células Dendríticas/patología , Regulación hacia Abajo/inmunología , Feto , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunofenotipificación , Mediadores de Inflamación/fisiología , Interleucina-4/fisiología , Lipopolisacáridos/farmacología , Microglía/patología , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/patologíaRESUMEN
Fas Ligand (FasL, CD178) is a cytokine that may be secreted or expressed as a transmembrane ligand at the cell surface, and induces apoptosis by binding to the "death receptor" Fas (CD95). Here, we show that Grb2, an SH3 domain-containing adaptor protein, binds to the proline-rich domain of FasL and regulates its cell surface expression. We found that knocking down Grb2 expression decreased the amount of FasL at the cell surface and increased the abundance of intracellular vesicles containing FasL. Furthermore, we showed that Grb2 acts as an adaptor for FasL to interact with adaptin beta, a molecule known to regulate trafficking. Our data reveal that Grb2 facilitates the association of FasL with adaptin beta, and promotes sorting of FasL to the cell surface. As FasL is a potent regulator of cell death, dynamic regulation of its cell surface localization is critical for controlling local tissue remodeling and inflammation.
Asunto(s)
Membrana Celular/metabolismo , Proteína Ligando Fas/metabolismo , Proteína Adaptadora GRB2/metabolismo , Células de Schwann/metabolismo , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Animales , Línea Celular , Proteína Ligando Fas/genética , Proteína Adaptadora GRB2/genética , Humanos , Ratones , Prolina/metabolismo , Estructura Terciaria de Proteína/genética , Transporte de Proteínas , Interferencia de ARN , Eliminación de SecuenciaRESUMEN
Members of the tumor necrosis factor receptor (TNFR) superfamily control cell fate determination, including cell death and differentiation. Fas (CD95) is the prototypical "death receptor" of the TNFR superfamily and signals apoptosis through well established pathways. In the adult nervous system, Fas induces apoptosis in the context of neuropathology such as stroke or amyotrophic lateral sclerosis. However, during nervous system development, Fas promotes neurite growth and branching. The molecular mechanisms underlying Fas-induced process formation and branching have remained unknown to date. Here, we define the molecular pathway linking Fas to process growth and branching in cell lines and in developing neurons. We describe a new cytoplasmic membrane proximal domain (MPD) that is essential for Fas-induced process growth and that is conserved in members of the TNFR superfamily. We show that the Fas MPD recruits ezrin, a molecule that links transmembrane proteins to the cytoskeleton, and activates the small GTPase Rac1. Deletion of the MPD, but not the death domain, abolished Rac1 activation and process growth. Furthermore, an ezrin-derived inhibitory peptide prevented Fas-induced neurite growth in primary neurons. Our results define a new domain, topologically and functionally distinct from the death domain, which regulates neuritogenesis via recruitment of ezrin and activation of Rac1.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica , Neuritas/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptor fas/química , Proteína de Unión al GTP rac1/metabolismo , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Modelos Biológicos , Neuronas/metabolismo , Estructura Terciaria de Proteína , Receptor fas/biosíntesisRESUMEN
Fas ligand (FasL) binds Fas (CD95) to induce apoptosis or activate other signaling pathways. In addition, FasL transduces bidirectional or 'reverse signals'. The intracellular domain of FasL contains consensus sequences for phosphorylation and an extended proline rich region, which regulate its surface expression through undetermined mechanism(s). Here, we used a proteomics approach to identify novel FasL interacting proteins in Schwann cells to investigate signaling through and trafficking of this protein in the nervous system. We identified two novel FasL interacting proteins, sorting nexin 18 and adaptin beta, as well as two proteins previously identified as FasL interacting proteins in T cells, PACSIN2 and PACSIN3. These proteins are all associated with endocytosis and trafficking, highlighting the tight regulation of cell surface expression of FasL in the nervous system.
Asunto(s)
Proteína Ligando Fas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Células de Schwann/metabolismo , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Proteína Ligando Fas/análisis , Proteína Ligando Fas/genética , Eliminación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteoma/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células de Schwann/citología , Espectrometría de Masas en Tándem , Transfección , Proteínas de Transporte Vesicular/metabolismo , Dominios Homologos srcRESUMEN
CD95 is the quintessential death receptor and, when it is bound by ligand, cells undergo apoptosis. Recent evidence suggests, however, that CD95 mediates not only apoptosis but also diverse nonapoptotic functions depending on the tissue and the conditions.
Asunto(s)
Apoptosis , Receptor fas/metabolismo , Animales , Proteína Ligando Fas/metabolismo , Humanos , Ratones , Neoplasias/inmunología , Neoplasias/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Neuroblastoma, a frequently occurring solid tumour in children, remains a therapeutic challenge as existing imaging tools are inadequate for proper and accurate diagnosis, resulting in treatment failures. Nanoparticles have recently been introduced to the field of cancer research and promise remarkable improvements in diagnostics, targeting and drug delivery. Among these nanoparticles, quantum dots (QDs) are highly appealing due to their manipulatable surfaces, yielding multifunctional QDs applicable in different biological models. The biocompatibility of these QDs, however, remains questionable. RESULTS: We show here that QD surface modifications with N-acetylcysteine (NAC) alter QD physical and biological properties. In human neuroblastoma (SH-SY5Y) cells, NAC modified QDs were internalized to a lesser extent and were less cytotoxic than unmodified QDs. Cytotoxicity was correlated with Fas upregulation on the surface of treated cells. Alongside the increased expression of Fas, QD treated cells had increased membrane lipid peroxidation, as measured by the fluorescent BODIPY-C11 dye. Moreover, peroxidized lipids were detected at the mitochondrial level, contributing to the impairment of mitochondrial functions as shown by the MTT reduction assay and imaged with confocal microscopy using the fluorescent JC-1 dye. CONCLUSION: QD core and surface compositions, as well as QD stability, all influence nanoparticle internalization and the consequent cytotoxicity. Cadmium telluride QD-induced toxicity involves the upregulation of the Fas receptor and lipid peroxidation, leading to impaired neuroblastoma cell functions. Further improvements of nanoparticles and our understanding of the underlying mechanisms of QD-toxicity are critical for the development of new nanotherapeutics or diagnostics in nano-oncology.
RESUMEN
Defects in the ubiquitin-proteasome system have been implicated in Parkinson's Disease (PD). Recently, a rat model of PD was developed using a synthetic proteasome inhibitor (PSI), (Z-lle-Glu(OtBu)-Ala-Leu-al). We attempted to transfer this model to mouse studies, where genetics can be more readily investigated due to the availability of genetically modified mice. We treated C57BL/6 (B6) mice with six intraperitoneal injections of 6 mg/kg PSI in 50 mul of 70% ethanol over a 2-week-period. We found significant decreases in nigrostriatal dopamine in PSI-treated mice compared with saline-treated mice. However, we observed similar decreases in the ethanol-treated vehicle control group. Administration of ethanol alone led to significant long-term alterations in dopamine levels. Ethanol significantly eclipses the effects of PSI in the dopamine system, and therefore is a confounding vehicle for this model.
Asunto(s)
Depresores del Sistema Nervioso Central/administración & dosificación , Inhibidores de Cisteína Proteinasa/toxicidad , Etanol/administración & dosificación , Oligopéptidos/toxicidad , Enfermedad de Parkinson/etiología , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Cromatografía Líquida de Alta Presión/métodos , Modelos Animales de Enfermedad , Dopamina/metabolismo , Ácido Homovanílico/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Although 1,25-dihydroxyvitamin D3 (1,25D3) and retinoic acid (RA) have distinct developmental and physiological roles, both regulate the cell cycle. We provide molecular and genomic evidence that their cognate nuclear receptors regulate common genes through everted repeat TGA(C/T)TPyN8PuG(G/T)TCA (ER8) response elements. ER8 motifs were found in the promoters of several target genes of 1,25D3 and/or RA. Notably, an element was characterized in the cyclin-dependent kinase (CDK) inhibitor p19ink4d gene, and 1,25D3- or RA-induced p19INK4D) expression. P19ink4d knockdown together with depletion of p27kip1, another CDK inhibitor regulated by 1,25D3 and RA, rendered cells resistant to ligand-induced growth arrest. Remarkably, p19INK4D-deficient cells showed increased autophagic cell death, which was markedly enhanced by 1,25D3, but not RA, and attenuated by loss of p27KIP1. These results show a limited crosstalk between 1,25D3 and RA signalling by means of overlapping nuclear receptor DNA binding specificities, and uncover a role for p19INK4D in control of cell survival.
Asunto(s)
Calcitriol/metabolismo , Transducción de Señal , Tretinoina/metabolismo , Elemento de Respuesta a la Vitamina D , Autofagia , Western Blotting , Calcitriol/farmacología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Regulación de la Expresión Génica , Histocitoquímica , Humanos , Lisosomas/enzimología , Microscopía Confocal , Regiones Promotoras Genéticas , ARN Interferente Pequeño/farmacología , Receptor Cross-Talk , Receptores de Calcitriol/metabolismo , Receptores de Ácido Retinoico/metabolismo , Tretinoina/farmacología , beta-Galactosidasa/metabolismoRESUMEN
Fas (CD95), a member of the tumor necrosis factor-receptor superfamily, has been studied extensively as a death-inducing receptor in the immune system. However, Fas is also widely expressed in a number of other tissues, including in neurons. Here, we report that defects in the Fas/Fas ligand system unexpectedly render mice highly susceptible to neural degeneration in a model of Parkinson's disease. We found that Fas-deficient lymphoproliferative mice develop a dramatic phenotype resembling clinical Parkinson's disease, characterized by extensive nigrostriatal degeneration accompanied by tremor, hypokinesia, and loss of motor coordination, when treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at a dose that causes no neural degeneration or behavioral impairment in WT mice. Mice with generalized lymphoproliferative disease, which express a mutated Fas ligand, display an intermediate phenotype between that of lymphoproliferative and WT mice. Moreover, Fas engagement directly protects neuronal cells from MPTP/1-methyl-4-phenylpyridinium ion toxicity in vitro. Our data show that decreased Fas expression renders dopaminergic neurons highly susceptible to degeneration in response to a Parkinson-causing neurotoxin. These findings constitute the first evidence for a neuroprotective role for Fas in vivo.
Asunto(s)
Trastornos Linfoproliferativos/metabolismo , Intoxicación por MPTP/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Transducción de Señal/fisiología , Regulación hacia Arriba , Receptor fas/metabolismo , Anciano , Animales , Encéfalo/patología , Cromatografía Líquida de Alta Presión , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Mutantes , Persona de Mediana EdadRESUMEN
Fas (also known as CD95), a member of the tumour-necrosis receptor factor family of 'death receptors', can induce apoptosis or, conversely, can deliver growth stimulatory signals. Here we report that crosslinking Fas on primary sensory neurons induces neurite growth through sustained activation of the extracellular-signal regulated kinase (ERK) pathway and the consequent upregulation of p35, a mediator of neurite outgrowth. In addition, functional recovery after sciatic nerve injury is delayed in Fas-deficient lpr mice and accelerated by local administration of antibodies against Fas, which indicates that Fas engagement may contribute to nerve regeneration in vivo. Our findings define a role for Fas as an inducer of both neurite growth in vitro and accelerated recovery after nerve injury in vivo.