ABSTRACT
The recent discovery of the Nogo family of myelin inhibitors and the Nogo-66 receptor opens up a very promising avenue for the development of therapeutic agents for treating spinal cord injury. Nogo-A, the largest member of the Nogo family, is a multidomain protein containing at least two regions responsible for inhibiting central nervous system (CNS) regeneration. So far, no structural information is available for Nogo-A or any of its structural domains. We have subcloned and expressed two Nogo-A fragments, namely the 182 residue Nogo-A(567-748) and the 66 residue Nogo-66 in Escherichia coli. CD and NMR characterization indicated that Nogo-A(567-748) was only partially structured while Nogo-66 was highly insoluble. Nogo-40, a truncated form of Nogo-66, has been previously shown to be a Nogo-66 receptor antagonist that is able to enhance CNS neuronal regeneration. Detailed NMR examinations revealed that a Nogo-40 peptide had intrinsic helix-forming propensity, even in an aqueous environment. The NMR structure of Nogo-40 was therefore determined in the presence of the helix-stabilizing solvent trifluoroethanol. The solution structure of Nogo-40 revealed two well-defined helices linked by an unstructured loop, representing the first structure of Nogo-66 receptor binding ligands. Our results provide the first structural insights into Nogo-A functional domains and may have implications in further designs of peptide mimetics that would enhance CNS neuronal regeneration.
Subject(s)
Growth Inhibitors/metabolism , Myelin Proteins/metabolism , Protein Structure, Tertiary , Amino Acid Sequence , Central Nervous System/physiology , Circular Dichroism , Growth Inhibitors/chemistry , Growth Inhibitors/genetics , Humans , Models, Molecular , Myelin Proteins/chemistry , Myelin Proteins/genetics , Nogo Proteins , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolismABSTRACT
Malignant gliomas are common and aggressive brain tumours associated with significant morbidity and mortality. We showed in this report that substratum adherence and migration by human U87MG glioma cells in culture were significantly attenuated by the extracellular domains of Nogo-A (Nogo-66) and the myelin-associated glycoprotein (MAG). U87MG cells contained significant amounts of endogenous Nogo-66 receptor (NgR), and treatment of the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) or NgR antibodies resulted in an increase in their ability to adhere to, or migrate through, Nogo-66- and MAG-coated substrates. Nogo-66 and MAG may therefore modulate glioma growth and migration by acting through the NgR, a phenomenon that has potential therapeutic implications.
Subject(s)
Cell Movement/drug effects , Gene Expression Regulation/drug effects , Myelin Proteins/metabolism , Myelin Proteins/pharmacology , Myelin-Associated Glycoprotein/pharmacology , Receptors, Cell Surface/metabolism , Antibodies/pharmacology , Blotting, Western/methods , Brain/metabolism , Cell Adhesion/drug effects , Cell Count , Cell Line , Dose-Response Relationship, Drug , GPI-Linked Proteins , Glioblastoma , Humans , Myelin Proteins/genetics , Nogo Proteins , Nogo Receptor 1 , Phosphatidylinositol Diacylglycerol-Lyase/pharmacology , Phosphoinositide Phospholipase C , Precipitin Tests/methods , Receptors, Cell Surface/genetics , Recombinant Proteins/pharmacology , Time Factors , Tumor Cells, CulturedABSTRACT
Axonal regeneration in the adult CNS is limited by the presence of several inhibitory proteins associated with myelin. Nogo-A, a myelin-associated inhibitor, is responsible for axonal outgrowth inhibition in vivo and in vitro. Here we study the onset and maturation of Nogo-A and Nogo receptor in the entorhino-hippocampal formation of developing and adult mice. We also provide evidence that Nogo-A does not inhibit embryonic hippocampal neurons, in contrast to other cell types such as cerebellar granule cells. Our results also show that Nogo and Nogo receptor mRNA are expressed in the adult by both principal and local-circuit hippocampal neurons, and that after lesion, Nogo-A is also transiently expressed by a subset of reactive astrocytes. Furthermore, we analyzed their regulation after kainic acid (KA) treatment and in response to the transection of the entorhino-hippocampal connection. We found that Nogo-A and Nogo receptor are differentially regulated after kainic acid or perforant pathway lesions. Lastly, we show that the regenerative potential of lesioned entorhino-hippocampal organotypic slice co-cultures is increased after blockage of Nogo-A with two IN-1 blocking antibodies. In conclusion, our results show that Nogo and its receptor might play key roles during development of hippocampal connections and that they are implicated in neuronal plasticity in the adult.
Subject(s)
Entorhinal Cortex/physiology , Hippocampus/physiology , Myelin Proteins/metabolism , Nerve Regeneration/physiology , Perforant Pathway/physiology , Receptors, Cell Surface/metabolism , Animals , Animals, Newborn , Antibodies/pharmacology , Astrocytes/cytology , Astrocytes/metabolism , Brain Injuries/chemically induced , Brain Injuries/physiopathology , COS Cells , Entorhinal Cortex/embryology , Entorhinal Cortex/injuries , Fetus , GPI-Linked Proteins , Gene Expression Regulation, Developmental/genetics , Gliosis/metabolism , Gliosis/physiopathology , Growth Cones/metabolism , Growth Cones/ultrastructure , Hippocampus/embryology , Hippocampus/injuries , Kainic Acid , Mice , Myelin Proteins/antagonists & inhibitors , Myelin Proteins/genetics , Neuronal Plasticity/physiology , Nogo Proteins , Nogo Receptor 1 , Perforant Pathway/embryology , Perforant Pathway/injuries , RNA, Messenger/metabolism , Receptors, Cell Surface/genetics , Receptors, Peptide/genetics , Receptors, Peptide/metabolismABSTRACT
The molecule Nogo has captured the imagination of many as a possible key player, and therefore therapeutic target, in the pathological settings of central nervous system (CNS) injury and degenerative pathology. Found in both glial cells and neurons, the endogenous, physiological role of Nogo is as yet unknown. Recently reported targeted disruption of the Nogo gene did not result in any obvious neuro-anatomical or neurological phenotype. Compared with wild-type mice, Nogo-deficient mice also did not exhibit a truly convincing enhancement in their ability to regenerate CNS neurons upon injury. Does the molecule have any important physiological function at all? Other recent discoveries of new interacting partners of Nogo at the mitochondria and the CNS paranode suggest intriguing links to the modulation of apoptosis and developmental organization or signalling at the axoglial junction.
Subject(s)
Cell Communication/physiology , Extracellular Fluid/metabolism , Intracellular Fluid/metabolism , Myelin Proteins/metabolism , Animals , Humans , Mitochondria/metabolism , Myelin Proteins/genetics , Nerve Regeneration/physiology , Neuroglia/metabolism , Neurons/metabolism , Nogo ProteinsABSTRACT
We report Nogo-A as an oligodendroglial component congregating and interacting with the Caspr-F3 complex at paranodes. However, its receptor Nogo-66 receptor (NgR) does not segregate to specific axonal domains. CHO cells cotransfected with Caspr and F3, but not with F3 alone, bound specifically to substrates coated with Nogo-66 peptide and GST-Nogo-66. Binding persisted even after phosphatidylinositol- specific phospholipase C (PI-PLC) removal of GPI-linked F3 from the cell surface, suggesting a direct interaction between Nogo-66 and Caspr. Both Nogo-A and Caspr co-immunoprecipitated with Kv1.1 and Kv1.2, and the developmental expression pattern of both paralleled compared with Kv1.1, implicating a transient interaction between Nogo-A-Caspr and K(+) channels at early stages of myelination. In pathological models that display paranodal junctional defects (EAE rats, and Shiverer and CGT(-/-) mice), distances between the paired labeling of K(+) channels were shortened significantly and their localization shifted toward paranodes, while paranodal Nogo-A congregation was markedly reduced. Our results demonstrate that Nogo-A interacts in trans with axonal Caspr at CNS paranodes, an interaction that may have a role in modulating axon-glial junction architecture and possibly K(+)-channel localization during development.
Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Central Nervous System/metabolism , Myelin Proteins/metabolism , Potassium Channels, Voltage-Gated , Potassium Channels/metabolism , Amino Acid Sequence , Animals , Axons/metabolism , CHO Cells , Contactins , Cricetinae , GPI-Linked Proteins , Immunohistochemistry , Kv1.1 Potassium Channel , Ligands , Mice , Mice, Knockout , Mice, Neurologic Mutants , Microscopy, Immunoelectron , Models, Neurological , Molecular Sequence Data , Myelin Proteins/genetics , Nerve Fibers, Myelinated/metabolism , Nogo Proteins , Nogo Receptor 1 , Oligodendroglia/metabolism , Rats , Rats, Wistar , Receptors, Cell Surface/metabolism , Receptors, Peptide/metabolism , TransfectionABSTRACT
The sprouting of neurites from a neuron represents a highly specialized form of cellular morphogenesis that must involve coordinated changes in two major cellular processes at two membrane locations: reorganization of the cytoskeleton and redirection of membrane traffic from the trans-Golgi network to the plasma membrane of the growth tip. How exactly are these two processes linked and how is spatial and temporal coordination achieved at the first instance of neurite sprouting? Recent advances may have already revealed some, if not most of the pieces in the puzzle. We discuss below, with some extrapolations, of what has recently come to light, and what more is needed to construct a coherent picture.
