RESUMEN
Dendritic cells (DC) reside at the center of the immunological universe, possessing the ability both to stimulate and inhibit various types of responses. Tolerogenic/regulatory DC with therapeutic properties can be generated through various means of manipulations in vitro and in vivo. Here we describe several attractive strategies for manipulation of DC using the novel technique of RNA interference (RNAi). Additionally, we overview some of our data regarding yet undescribed characteristics of RNAi in DC such as specific transfection strategies, persistence of gene silencing, and multi-gene silencing. The advantages of using RNAi for DC genetic manipulation gives rise to the promise of generating tailor-made DC that can be used effectively to treat a variety of immunologically mediated diseases.
Asunto(s)
Células Dendríticas/inmunología , Interferencia de ARN/inmunología , ARN Interferente Pequeño/inmunología , Animales , Células Dendríticas/trasplante , Humanos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Inmunoterapia Adoptiva , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
p190RhoGEF is a large multi-functional protein with guanine nucleotide exchange (GEF) activity. The C-terminal region of p190RhoGEF is a highly interactive domain that binds multiple factors, including proteins with anti-apoptotic activities. We now report that transfection of EGFP-tagged p190RhoGEF protects Neuro 2a cells from stress-induced apoptosis and that anti-apoptotic activity is localized to cytoplasmic retention sequences (CRS-1 and CRS-2) in the C-terminal region of p190RhoGEF. Cytoplasmic retention is conferred to an EGFP fluorescent marker when fused to either CRS-1 or CRS-2. Both cytoplasmic retention and anti-apoptotic activity are lost by deleting CRS-1 and CRS-2 in the p190RhoGEF sequence and can be recovered by restoring either CRS-1 or CRS-2 to the EGFP-tagged sequence. Since the CRS-1 and CRS-2 contain the JIP-1 and 14-3-3 binding sites, we propose that anti-apoptotic activity may be conferred by the binding of p190RhoGEF to JIP-1 or 14-3-3, possibly by altering their interactive properties or nucleocytoplasmic movements. Taken together, our findings support a model whereby multiple interactions of p190RhoGEF confer homeostatic properties to differentiated neurons and may link neuronal homeostasis to the regulation of NF-L expression.
Asunto(s)
Apoptosis/fisiología , Citoplasma/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Luminiscentes/metabolismo , Proteínas Nucleares/metabolismo , Animales , Sitios de Unión , Western Blotting , Proteínas Portadoras/metabolismo , Agregación Celular , Muerte Celular , Línea Celular , Proteínas de Unión al ADN , Proteínas Activadoras de GTPasa , Glutatión Transferasa/genética , Proteínas Fluorescentes Verdes , Etiquetado Corte-Fin in Situ/métodos , Microscopía Confocal , Fragmentos de Péptidos/metabolismo , Pruebas de Precipitina/métodos , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras , Homología de Secuencia de Aminoácido , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons and causes progressive muscle weakness and atrophy. The etiology and pathogenesis of ALS are largely unknown and no effective treatment is presently available. About 10% of patients have the familial or inherited form of the disease (fALS), among which 20% is linked to mutations with Cu(2+)/Zn(2+) superoxide dismutase (mSOD1). Transgenic animals expressing human mSOD1 are excellent models for understanding not only fALS but sporadic ALS as well. Pathological features in both ALS patients and mSOD1 transgenic animals' spinal cords share commonalties including the accumulation of misfolded protein inclusions. Recent proteomic investigations on ALS animal models have discovered alterations in protein expression, protein-protein interactions and post-translational modifications. These efforts have revealed aspects of potential pathogenic mechanisms and identified probable therapeutic targets. The present review summarizes the major findings of proteomics studies performed on the mSOD1 mice with particular emphasis on the spinal cord proteome. These results are compared with those reported using cell cultures or specimens obtained from ALS patients. The convergence of pathogenic processes on protein chaperone function, and its relationship to protein degradation, metabolic dysfunction and oxidative signaling events is discussed.
RESUMEN
We have previously reported that altered stability of low molecular weight neurofilament (NFL) mRNA in lumbar spinal cord homogenates in amyotrophic lateral sclerosis (ALS) is associated with altered expression of trans-acting 3' UTR mRNA binding proteins. We have identified two hexanucleotide motifs as the main cis elements and, using LC/MS/MS of peptide digests of NFL 3' UTR interacting proteins from human spinal cord, observed that 14-3-3 proteins interact with these motifs. 14-3-3 beta, zeta, tau, gamma, and eta isoforms were found to be expressed in human spinal cord. Each isoform was expressed in vitro and shown to interact with NFL 3' UTR mRNA. Mutation of one or both motifs resulted in decreased 14-3-3 interaction, changes in predicted mRNA structure or alteration in stability of the mRNA. These data show a novel interaction for 14-3-3 with NFL mRNA, and suggests that 14-3-3 may play a role in regulating NFL mRNA stability.
Asunto(s)
Proteínas 14-3-3/metabolismo , Regiones no Traducidas 3'/metabolismo , Neuronas Motoras/metabolismo , Proteínas de Neurofilamentos/genética , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Regiones no Traducidas 3'/genética , Secuencias de Aminoácidos/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Sitios de Unión/genética , Línea Celular , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patología , Humanos , Mutación/genética , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Proteínas de Neurofilamentos/biosíntesis , Unión Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Médula Espinal/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal disease of unknown etiology. Mutations in copper/zinc superoxide dismutase (SOD1) are the most commonly associated genetic abnormality. Given that SOD1 is ubiquitously expressed, the exclusive vulnerability of motor neurons is one of the most puzzling issues in ALS research. We here report that wild-type SOD1 mRNA forms ribonucleoprotein (RNP) complexes with protein homogenates of neuronal tissue but not with homogenates of non-neuronal tissues. 3' Untranslated region of SOD1 mRNA-dependent RNP complexes functioned to stabilize SOD1 mRNA. Moreover, SOD1 mRNAs harboring ALS-associated mutations, including silent mutations, were deficient in forming RNP complexes. In contrast, SOD1 mRNAs harboring artificial mutations, not known to be associated with ALS, demonstrated preserved RNP complex formation. This paper reports RNP complex formation on SOD1 mRNA as a neuronal tissue-specific and ALS-associated mutation sensitive feature.
Asunto(s)
Enfermedad de la Neurona Motora/genética , Mutación , ARN Mensajero/genética , Superóxido Dismutasa/genética , Humanos , Enfermedad de la Neurona Motora/enzimología , Neuronas , ARN/genética , ARN/aislamiento & purificación , Sondas ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Transcripción GenéticaRESUMEN
The mechanism by which mutated copper-zinc superoxide dismutase (SOD1) causes familial amyotrophic lateral sclerosis is believed to involve an adverse gain of function, independent of the physiological antioxidant enzymatic properties of SOD1. In this study, we have observed that mutant SOD1 (G41S, G85A, and G93A) but not the wild type significantly reduced the stability of the low molecular weight neurofilament mRNA in a dosage-dependent manner. We have also demonstrated that mutant SOD1 but not the wild type bound directly to the neurofilament mRNA 3'-untranslated region and that the binding was necessary to induce mRNA destabilization. These observations provide an explanation for a novel gain of function in which mutant SOD1 expression in motor neurons alters an intermediate filament protein expression.
Asunto(s)
Proteínas de Neurofilamentos/genética , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismo , Línea Celular , Humanos , Neuronas Motoras/metabolismo , Mutación , Proteínas de Neurofilamentos/metabolismo , Unión Proteica , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
We have examined the steady-state levels of intermediate filament mRNA in amyotrophic lateral sclerosis using the RNAse protection assay (NFL, NFM, NFH; corrected against GAPDH) or by PCR (peripherin, alpha-internexin, nestin, and vimentin; corrected against beta-actin). Significant elevations of NFL and peripherin mRNA levels were observed within the ALS cervical and lumbar spinal cord, with all other IF mRNA levels being comparable between control and ALS cases. These findings suggest that disturbances in both NFL and peripherin expression, independently known to contribute to the generation of motor neuron dysfunction in transgenic mice, are evident in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Esclerosis Amiotrófica Lateral/genética , Encéfalo/metabolismo , Humanos , Proteínas de Filamentos Intermediarios/genética , Ensayos de Protección de Nucleasas , ARN Mensajero/metabolismo , Médula Espinal/metabolismoRESUMEN
Neurofilament (NF) aggregates in motor neurons are a key neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously observed an alteration in the stoichiometry of NF subunit steady state mRNA levels in ALS spinal motor neurons using in situ hybridization and proposed that this led to aggregate formation. We have now examined the levels of NF mRNA in whole tissue homogenates of spinal cord using the RNase protection assay and real time reverse transcriptase-PCR and observed significant elevations of NF mRNA level in ALS. Compared with age-matched control, we observed a greater stability of heterogeneously expressed NFL mRNA in the presence of ALS spinal cord homogenates. Heat denaturing or protease K digestion of the control homogenates increased the stability of the NFL mRNA to levels observed in ALS homogenate. Increased NFL mRNA stability was also induced by increasing the percentage of ALS homogenate in an admixture of control and ALS homogenates. These observations suggest the presence of trans-acting NFL mRNA-destabilizing elements in control but not in ALS spinal cord homogenates. This was confirmed in gel retardation assays. We also observed that the destabilizing elements interact with the 3'-untranslated region of NFL mRNA. These findings suggest that the trans-acting NFL-destabilizing elements are selectively suppressed in ALS homogenates, resulting in an increased stability and level of NFL mRNA.