RESUMEN
Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease.
Asunto(s)
Autofagia/efectos de los fármacos , Calpaína/antagonistas & inhibidores , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Péptidos/metabolismo , Animales , Proteínas de Unión al Calcio/biosíntesis , Calpaína/genética , Calpaína/metabolismo , Modelos Animales de Enfermedad , Drosophila , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/terapia , Endogamia , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
BACKGROUND: Mast cells (MCs) are the classical mediators of allergy, however, their importance in the development of innate and adaptive immune responses is increasingly being recognized. Herein, the present MC literature is summarized, with particular focus on studies of MCs in the endometrium and myometrium, and their involvement in fertility, implantation, pregnancy and labour. METHODS: Recent developments in MC biology were identified by systematic searches of PubMed, Medline and Google Scholar from 2000 to November 2009. To specifically examine the role of MCs in fertility and pregnancy, we then performed a systematic review of English literature cited in the PubMed, Medline and Google Scholar databases, but extended the search period, from 1980 to January 2010 RESULTS: MCs can respond to immunoglobulin E-independent innate immune stimuli and are present within the endometrium, with activation and release of mediators occurring prior to menstruation and in association with endometriosis. With respect to pregnancy, MCs are redundant during blastocyst implantation and although their mediators can induce myometrial contractility, there is no epidemiological link of preterm birth with allergy, suggesting a non-essential role or robust regulation. In males, MCs are present in the testes and are increased in oligo- and azoospermia, with MC mediators directly suppressing sperm motility in a potentially reversible manner. CONCLUSIONS: MCs are prevalent in the female and male reproductive tract. However, whether MCs are absolutely required for a successful pregnancy or are fundamental to reproductive pathology, and thereby a therapeutic target, remains to be determined.
Asunto(s)
Trabajo de Parto/fisiología , Mastocitos/fisiología , Reproducción/fisiología , Movimiento Celular , Implantación del Embrión/fisiología , Endometrio/citología , Femenino , Humanos , Hipersensibilidad/complicaciones , Infertilidad Masculina/patología , Masculino , Modelos Biológicos , Miometrio/citología , Placenta/citología , Embarazo , Complicaciones del Embarazo/inmunología , Complicaciones del Embarazo/patologíaRESUMEN
The present study examines the temporal dynamics of macrophage activation marker expression in response to variations in stimulation. We demonstrate that markers can be categorized as 'early' (expressed most abundantly at 6 h post-stimulation) or 'late' (expressed at 24 h post-stimulation). Thus nos2 and p40 (IL-12/IL-23) are early markers of innate and classical activation, while dectin-1 and mrc-1 are early markers and fizz1 (found in inflammatory zone-1) and ym1 are late markers of alternative activation. Furthermore, argI is a late marker of both innate and alternative activation. The ability of interferon (IFN)-gamma to alter these activation markers was studied at both the protein level and gene level. As reported previously, IFN-gamma was able to drive macrophages towards the classical phenotype by enhancing nos2 gene expression and enzyme activity and p40 (IL-12/IL-23) gene expression in lipopolysaccharide (LPS)-stimulated macrophages. IFN-gamma antagonized alternative macrophage activation, as evident by reduced expression of dectin-1, mrc-1, fizz1 and ym1 mRNA transcripts. In addition, IFN-gamma antagonized arginase activity irrespective of whether macrophages were activated innately or alternatively. Our data explain some apparent contradictions in the literature, demonstrate temporal plasticity in macrophage activation states and define for the first time 'early' and 'late' markers associated with anti-microbial/inflammatory and wound healing responses, respectively.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Mediadores de Inflamación/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Cicatrización de Heridas/inmunología , Animales , Antivirales/farmacología , Arginasa/biosíntesis , Arginasa/inmunología , Biomarcadores/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/inmunología , Interferón gamma/farmacología , Subunidad p40 de la Interleucina-12/biosíntesis , Subunidad p40 de la Interleucina-12/inmunología , Lectinas/biosíntesis , Lectinas/inmunología , Lectinas Tipo C , Activación de Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/inmunología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/inmunología , Cicatrización de Heridas/efectos de los fármacos , beta-N-Acetilhexosaminidasas/biosíntesis , beta-N-Acetilhexosaminidasas/inmunologíaRESUMEN
Free radical damage has been implicated in the pathophysiology of motor neurone disease (MND); mutations have been identified in the gene encoding Cu/Zn superoxide dismutase (SOD1). There is evidence that glial cell dysfunction may contribute to motor neurone injury, but the exact role of glial cells in MND has yet to be established. The aim of this study was to determine whether expression of mutant SOD1 affects the response of glia to oxidative stress. Stable C6 glioma cells expressing mutant SOD1 and cortical astrocyte cultures from G93A-SOD1 transgenic mice were exposed to: xanthine/xanthine oxidase; hydrogen peroxide; A23187 and 3-morpholinosydonimine. Cell viability was measured using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Neither C6 glioma cells nor cortical astrocytes expressing mutant SOD1 were more susceptible to any of the free radical generating systems compared to control cells. These results suggest that astrocytes are resistant to the toxic effects of mutant SOD1 widely reported for neuronal cells.
Asunto(s)
Astrocitos/enzimología , Enfermedad de la Neurona Motora/enzimología , Enfermedad de la Neurona Motora/genética , Mutación/fisiología , Superóxido Dismutasa/genética , Células Tumorales Cultivadas/enzimología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Sistema Nervioso Central/enzimología , Sistema Nervioso Central/patología , Sistema Nervioso Central/fisiopatología , Radicales Libres/metabolismo , Radicales Libres/farmacología , Glioma , Ratones , Ratones Transgénicos , Enfermedad de la Neurona Motora/fisiopatología , Neuronas Motoras/enzimología , Neuronas Motoras/patología , Mutación/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Superóxido Dismutasa/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patologíaRESUMEN
Modification of the growth conditions of NSC-34 mouse neuroblastoma x motor neurone cells by serum depletion promotes the expression of functional glutamate receptors as the cells mature into a form that bears the phenotypic characterisation of motor neurones. Immunocytochemical studies demonstrated the presence of the glutamate receptor proteins NMDAR1, NMDAR2A/B, GluR1, GluR2, GluR2/3, GluR4, GluR6/7, and KA2. Toxicity assays using cell counting techniques demonstrated a mild but significant cell death (approximately 30%, p < 0.01) following a 24-h exposure to 1 mM glutamate that could be prevented by the presence of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (10 microM) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (1 microM). As an indication of glutamate receptor functional activity a novel approach was used to detect the production of free radicals following stimulation with glutamate receptor agonists. The release of superoxide free radicals was detected using a micro-electrochemical sensor following addition of glutamate receptor agonists to the cell bathing solution. Alterations in intracellular calcium concentrations were examined using fura-2 imaging. Exposure of the differentiated NSC-34 cells to glutamate leads to an increase in intracellular calcium concentrations that is prevented by the presence of glutamate receptor antagonists. The motor neurone origin of these cells makes them particularly useful for investigating the potential role of glutamatergic toxicity in motor neurone degeneration.