RESUMEN
The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.
Asunto(s)
Autoanticuerpos/biosíntesis , Autoinmunidad , Glomerulonefritis Membranoproliferativa/genética , Factores de Intercambio de Guanina Nucleótido/genética , Animales , Autoanticuerpos/sangre , Proteína C9orf72 , Citocinas/sangre , Femenino , Glomerulonefritis Membranoproliferativa/sangre , Glomerulonefritis Membranoproliferativa/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos , Tejido Linfoide/patología , Macrófagos/inmunología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/inmunología , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
The vasculature of the CNS is structurally and functionally distinct from that of other organ systems and is particularly prone to developmental abnormalities and hemorrhage. Although other embryonic tissues undergo primary vascularization, the developing nervous system is unique in that it is secondarily vascularized by sprouting angiogenesis from a surrounding perineural plexus. This sprouting angiogenesis requires the TGF-ß and Wnt pathways because ablation of these pathways results in aberrant sprouting and hemorrhage. We have genetically deleted Gpr124, a member of the large family of long N-terminal group B G protein-coupled receptors, few members of which have identified ligands or well-defined biologic functions in mammals. We show that, in the developing CNS, Gpr124 is specifically expressed in the vasculature and is absolutely required for proper angiogenic sprouting into the developing neural tube. Embryos lacking Gpr124 exhibit vascular defects characterized by delayed vascular penetration, formation of pathological glomeruloid tufts within the CNS, and hemorrhage. In addition, they display defects in palate and lung development, two processes in which TGF-ß and/or Wnt pathways also play important roles. We also show that TGF-ß stimulates Gpr124 expression, and ablation of Gpr124 results in perturbed TGF-ß pathway activation, suggesting roles for Gpr124 in modulating TGF-ß signaling. These results represent a unique function attributed to a long N-terminal group B-type G protein-coupled receptor in a mammalian system.
Asunto(s)
Sistema Nervioso Central/irrigación sanguínea , Sistema Nervioso Central/embriología , Neovascularización Fisiológica/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Embrión de Mamíferos , Ingeniería Genética , Técnicas Histológicas , Inmunohistoquímica , Hibridación in Situ , Pulmón/embriología , Pulmón/metabolismo , Ratones , Análisis por Micromatrices , Hueso Paladar/embriología , Hueso Paladar/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis.
Asunto(s)
Células Madre Embrionarias/enzimología , Células Madre Embrionarias/patología , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/enzimología , Neovascularización Patológica , Proteínas Tirosina Quinasas Receptoras/fisiología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/fisiología , Angiopoyetinas/antagonistas & inhibidores , Animales , Diferenciación Celular , Línea Celular , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Células Endoteliales/patología , Ratones , Ratones SCID , Neoplasias Experimentales/etiología , Receptor TIE-2 , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/deficiencia , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Teratoma/irrigación sanguínea , Teratoma/enzimología , Teratoma/etiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Nogo-A is a potent neurite growth inhibitor in vitro and plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.
Asunto(s)
Proteínas de la Mielina/fisiología , Neuritas/fisiología , Células 3T3 , Animales , Axones/efectos de los fármacos , Axones/fisiología , Sitios de Unión/fisiología , Biotinilación , Química Encefálica , Células CHO , Adhesión Celular , Membrana Celular/química , Membrana Celular/metabolismo , Corteza Cerebral/química , Corteza Cerebral/metabolismo , Embrión de Pollo , Cricetinae , Fibroblastos/metabolismo , Proteínas Ligadas a GPI , Ratones , Datos de Secuencia Molecular , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Proteínas Nogo , Receptor Nogo 1 , Oligodendroglía/metabolismo , Unión Proteica/fisiología , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Ratas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Eliminación de SecuenciaRESUMEN
BACKGROUND: In this study we describe a new approach for expression cloning of receptors. METHODS: Our approach was based on highly efficient transfer of retroviral cDNA libraries into target cells and detection of receptor-ligand interaction with the use of an antibody directed against an epitope tag on recombinant ligands. Detection of the complex and isolation of receptor-transduced cells were achieved by flow cytometry and rare event high-speed cell sorting. Recovery of the cDNA coding for the receptor(s) was achieved by polymerase chain reaction. RESULTS: As a proof-of-concept study we set out to clone the receptor for B-lymphocyte stimulator protein (BlyS), not known at the start of the project but reported while this work was in progress. First, we detected binding of epitope-tagged BlyS to IM9 cells. Second, human T-lymphoblasts (CEM cells), which do not bind BlyS, were transduced with a retroviral cDNA library generated from IM9 cells. Transduced CEM cells binding epitope-tagged BlyS protein were identified by flow cytometry. After three sequential rounds of cell sorting, transduced CEM cell populations with high binding capacity for BlyS were identified. To determine the cDNAs conferring binding to the transduced CEM cells, the integrated proviral DNAs were amplified by polymerase chain reaction and analyzed by DNA sequencing. Rescued cDNAs contained Transmembrane Activator and calcium-modulator and cyclophilin ligand (CAML) Interactor (TACI) and B-Cell Maturation factor (BCMA) sequences, representing two published receptors of BlyS. CONCLUSIONS: Our data demonstrated that flow cytometry and high-speed cell sorting combined with transduction of retroviral cDNA libraries and binding of epitope-tagged orphan ligands as a selectable phenotype can be used efficiently for expression cloning of receptors. Of particular interest was our finding that apparently it is not necessary to purify the ligand but that conditioned medium containing the ligand can be used instead. Thus we concluded that our approach shortens the time to identify receptors for many orphan ligands and helps to exploit these receptors as drug targets.