ABSTRACT
Presynaptic terminals contain highly organized subcellular structures to facilitate neurotransmitter release. In C. elegans, the typical presynaptic terminal has an electron-dense active zone surrounded by synaptic vesicles. Loss-of-function mutations in the rpm-1 gene result in abnormally structured presynaptic terminals in GABAergic neuromuscular junctions (NMJs), most often manifested as a single presynaptic terminal containing multiple active zones. The RPM-1 protein has an RCC1-like guanine nucleotide exchange factor (GEF) domain and a RING-H2 finger. RPM-1 is most similar to the Drosophila presynaptic protein Highwire (HIW) and the mammalian Myc binding protein Pam. RPM-1 is localized to the presynaptic region independent of synaptic vesicles and functions cell autonomously. The temperature-sensitive period of rpm-1 coincides with the time of synaptogenesis. rpm-1 may regulate the spatial arrangement, or restrict the formation, of presynaptic structures.
Subject(s)
Adaptor Proteins, Signal Transducing , Caenorhabditis elegans Proteins , Caenorhabditis elegans/metabolism , Drosophila Proteins , Guanine Nucleotide Exchange Factors/physiology , Mixed Function Oxygenases , Presynaptic Terminals/physiology , Amino Acid Sequence , Animals , Biomarkers , Carrier Proteins/genetics , Drosophila/metabolism , Green Fluorescent Proteins , Guanine Nucleotide Exchange Factors/genetics , Indicators and Reagents/pharmacokinetics , Luminescent Proteins/pharmacokinetics , Molecular Sequence Data , Mutation/genetics , Mutation/physiology , Nerve Tissue Proteins/genetics , Neurons/physiology , Presynaptic Terminals/metabolism , Receptors, GABA/metabolism , Synapses/metabolism , Synapses/physiology , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructure , Tissue Distribution , Ubiquitin-Protein Ligases , Zinc Fingers/geneticsABSTRACT
Ionotropic GABA receptors generally require the products of three subunit genes. By contrast, the GABA receptor needed for locomotion in Caenorhabditis elegans requires only the unc-49 gene. We cloned unc-49 and demonstrated that it possesses an unusual overlapping gene structure. unc-49 contains a single copy of a GABA receptor N terminus, followed by three tandem copies of a GABA receptor C terminus. Using a single promoter, unc-49 generates three distinct GABAA receptor-like subunits by splicing the N terminus to each of the three C-terminal repeats. This organization suggests that the three UNC-49 subunits (UNC-49A, UNC-49B, and UNC-49C) are coordinately rescued and therefore might coassemble to form a heteromultimeric GABA receptor. Surprisingly, only UNC-49B and UNC-49C are expressed at high levels, whereas UNC-49A expression is barely detectable. Green fluorescent protein-tagged UNC-49B and UNC-49C subunits are coexpressed in muscle cells and are colocalized to synaptic regions. UNC-49B and UNC-49C also coassemble efficiently in Xenopus oocytes and HEK-293 cells to form a heteromeric GABA receptor. Together these data argue that UNC-49B and UNC-49C coassemble at the C. elegans neuromuscular junction. Thus, C. elegans is able to encode a heteromeric GABA receptor with a single locus.
Subject(s)
Caenorhabditis elegans/genetics , Genes, Helminth , Receptors, GABA/genetics , Receptors, GABA/metabolism , Alternative Splicing , Amino Acid Sequence , Animals , Caenorhabditis elegans/chemistry , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Cell Line , Cloning, Molecular , Genes, Essential/genetics , Humans , Molecular Sequence Data , Muscles/cytology , Muscles/metabolism , Neuromuscular Junction/chemistry , Neuromuscular Junction/cytology , Neuromuscular Junction/metabolism , Oocytes/metabolism , Phenotype , Phylogeny , Protein Isoforms/analysis , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, GABA/analysis , Receptors, GABA/chemistry , Structure-Activity Relationship , Synapses/chemistry , Synapses/metabolism , Tandem Repeat Sequences/genetics , Tandem Repeat Sequences/physiology , Xenopus laevisABSTRACT
Oncostatin M (OM), a member of the IL-6 gene family, stimulates a variety of functions implicated in wound repair. Transgenic mice that express this cytokine in islet beta-cells develop a connective tissue disorder that typifies excessive healing with severe fibrosis and lymphocytic infiltration. To compare this phenotype with the normal progression of connective tissue disease, we measured the expression patterns of genes encoding proinflammatory cytokines, fibrogenic cytokines, and ECM components by in situ hybridization. To test whether the OM effect was caused by its ability to regulate IL-6, we crossed the OM transgene into IL-6-deficient mice. Our data suggest that the fibrosis in these animals is not a secondary consequence of inflammation, or IL-6 expression, but is a direct effect by OM on extracellular matrix production. In a separate experiment, we observed that OM could regulate vasoactive intestinal peptide gene expression in the neurons that innervate the transgenic pancreas. This nerve healing response, in combination with its fibrogenic activity, suggests that OM functions downstream of inflammation in the wound repair cascade. These transgenic mice represent a useful model in which the fibroproliferative phase of connective tissue disease is uncoupled from inflammation.
Subject(s)
Connective Tissue Diseases/metabolism , Extracellular Matrix Proteins/genetics , Interleukin-6/physiology , Pancreas/pathology , Peptides/physiology , Animals , Animals, Newborn , Cattle , Connective Tissue Diseases/pathology , Crosses, Genetic , Cytokines/genetics , Cytokines/physiology , Disease Models, Animal , Fibrosis , Gene Expression Regulation , Growth Substances/genetics , Interleukin-6/genetics , Islets of Langerhans/metabolism , Leukocytes, Mononuclear , Mice , Mice, Transgenic , Oncostatin M , Pancreas/immunology , Pancreas/innervation , Pancreas/metabolism , RNA, Messenger/analysis , Sympathetic Nervous System , Transgenes , Vasoactive Intestinal Peptide/genetics , Wound Healing/physiologyABSTRACT
The neurodegenerative disease amyotrophic lateral sclerosis (ALS) results from the progressive loss of motoneurons, leading to death in a few years. Ciliary neurotrophic factor (CNTF), which decreases naturally occurring and axotomy-induced cell death, may result in slowing of motoneuron loss and has been evaluated as a treatment for ALS. Effective administration of this protein to motoneurons may be hampered by the exceedingly short half-life of CNTF, and the inability to deliver effective concentration into the central nervous system after systemic administration in vivo. The constitutive release of CNTF from genetically engineered cells may represent a solution to this delivery problem. In this work, baby hamster kidney (BHK) cells stably tranfected with a chimeric plasmid construct containing the gene for human or mouse CNTF were encapsulated in polymer fibers, which prevents immune rejection and allow long-term survival of the transplanted cells. In vitro bioassays show that the encapsulated transfected cells release bioactive CNTF. In vivo, systemic delivery of human and mouse CNTF from encapsulated cells was observed to rescue 26 and 27% more facial motoneurons, respectively, as compared to capsules containing parent BHK cells 1 wk postaxotomy in neonatal rats. With local application of CNTF on the nerve stump and by systemic delivery through repeated subcutaneous injections, 15 and 13% more rescue effects were observed. These data illustrate the potential of using encapsulated genetically engineered cells to continuously release CNTF to slow down motoneuron degeneration following axotomy and suggest that encapsulated cell delivery of neurotrophic factors may provide a general method for effective administration of therapeutic proteins for the treatment of neurodegenerative diseases.
Subject(s)
Amyotrophic Lateral Sclerosis/therapy , Axons/ultrastructure , Drug Delivery Systems/methods , Motor Neurons/cytology , Nerve Tissue Proteins/metabolism , Animals , Blotting, Northern , Cell Death , Cells, Cultured , Chick Embryo , Ciliary Neurotrophic Factor , Cricetinae , Enzyme-Linked Immunosorbent Assay , Genetic Engineering/methods , Humans , Mice , Motor Neurons/transplantation , Polymers , Rats , Rats, Sprague-DawleyABSTRACT
Porphyromonas gingivalis is a gram-negative bacterium that is associated with periodontitis. It has been hypothesized that destruction of bone and periodontal connective tissue is associated with colonization of the subgingival crevicular space by P. gingivalis, although how these bacteria overcome innate host defenses is largely unknown. To examine the early cellular and molecular events of P. gingivalis interaction with host tissues, we compared lipopolysaccharide (LPS) isolated from this bacterium with Escherichia coli LPS, a potent inflammatory mediator, in a mouse model of acute inflammation. In these studies, mice were given intramuscular injections of either P. gingivalis LPS or E. coli LPS and then sacrificed after 4 h. Reverse transcriptase-PCR analysis showed that expression of mRNAs for E- and P-selectins was higher in E. coli LPS-injected muscles than in P. gingivalis LPS-injected or control phosphate-buffered-saline-injected muscles. Similarly, monocyte chemotactic protein 1 and fibroblast-induced cytokine mRNAs were expressed in E. coli LPS-injected muscles whereas their expression was reduced or absent in P. gingivalis LPS-injected samples. These results were confirmed by in situ hybridization whereby stronger hybridization for selectin mRNAs was observed in the endothelium of capillaries from E. coli LPS-injected samples than in that from P. gingivalis LPS-injected muscles. In addition, many monocytes expressing monocyte chemotactic protein 1 mRNA and polymorphonuclear leukocytes expressing fibroblast-induced cytokine mRNA were observed in E. coli LPS-injected muscles whereas only a few cells were identified in P. gingivalis LPS-injected muscles. These results demonstrate that compared with E. coli, P. gingivalis has a low biologically reactive LPS as measured by its weak activation of inflammation. This may allow P. gingivalis to evade innate host defense mechanisms, resulting in colonization and chronic disease.
Subject(s)
Inflammation/immunology , Lipopolysaccharides/immunology , Porphyromonas gingivalis/immunology , Animals , Base Sequence , Chemokine CCL2/genetics , Cytokines/genetics , Disease Models, Animal , E-Selectin/genetics , Escherichia coli/pathogenicity , Inflammation/microbiology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , P-Selectin/genetics , Polymerase Chain Reaction , Porphyromonas gingivalis/pathogenicity , RNA, Messenger/analysisABSTRACT
Leukemia inhibitory factor (LIF) is a cytokine growth factor that induces rat sympathetic neurons to switch their neurotransmitter phenotype from noradrenergic to cholinergic in vitro. To test whether LIF can influence neuronal differentiation in vivo, we generated transgenic mice that expressed LIF in pancreatic islets under the control of the insulin promoter and evaluated the neurotransmitter phenotype of the pancreatic sympathetic innervation. We also used the insulin promoter to coexpress nerve growth factor in the islets, which greatly increased the density of sympathetic innervation and facilitated analysis of the effects of LIF. Our data demonstrate that tyrosine hydroxylase and catecholamines declined and choline acetyltransferase increased in response to LIF. We conclude that LIF can induce neurotransmitter switching of sympathetic neurons in vivo.