Myelination of axons in the vertebrate nervous system is essential for the fast propagation of action potentials. The multilamellar myelin sheath is a specialised, polarised, lipid-rich domain of the glial cell plasma membrane, synthesised by oligodendrocytes in the CNS and Schwann cells in the PNS. Loss of myelin, results in paralysis. Our aim is to define the molecules of glial precursor cells regulating the migration to and interaction with myelination-competent neurones and to elucidate the cell biological principles underlying the synthesis of myelin. The mechanism of sorting and compartmentation of proteins and lipids in myelinating glia, for example via lipid rafts, is also a central topic. These topics are additionally of clinical relevance in the context of de- and dysmyelinating diseases (e.g. Multiple Sclerosis), the migration of neural tumours and the missorting of glial proteins and lipids in the pathology of human disease. Recent work is also addressing the interaction of glial progenitor cells with neurones at synapses.
The AN2/NG2 Glycoprotein in Development and Disease
We have recently shown that the AN2 glycoprotein is the mouse homologue of human and rat NG2. It is expressed by migratory oligodendroglial and Schwann cell precursors and is down-regulated as these cells mature. It is also expressed by synaptic glia and may play a role in regulating synpatic activity and network properties. A subset of patients with the demyelinating disease Multiple Sclerosis, synthesise antibodies against NG2: this may contribute to the poor repair seen in these patients as the precursor cells responsible for remyelination are attacked. Additionally, the antibodies may be interfering with synaptic function. We have cloned mouse NG2 and generated fusion proteins and specific antisera for the different regions of the molecule. These provide tools to investigate the function of the molecule. We have recently shown that the C-terminus of NG2 binds to the PDZ protein GRIP and thus forms a complex with AMPA receptors in synaptic glia and immature oligodendrocytes. We have also identified a link between the cytoplasmic region of NG2 and the cytoskeleton which may play a role in cell and cell process movement and cell migration.
Oligodendroglia sort GPI-anchored adhesion molecules associated with Signalling Complexes into lipid rafts in the early phases of Glial-Axon recognition
In maturing oligodendrocytes, the glycosylphosphatidylinositol-anchored proteins F3 and NCAM 120 associate with the intracellular tyrosine kinase Fyn and the myelin lipids cholesterol and glycosphingolipids in detergent-resistant "rafts". The activity of Fyn is highest during myelination. These complexes may thus couple adhesion molecules to signal transduction cascades inside the glial cell whose activation stimulates the myelination programme. Ligation of such adhesion molecules by axonal ligands may thus induce cytoskeletal changes leading to the wrapping of axons and the laying down of the multilamellar sheath. We have shown that in oligodendrocytes Fyn associates with the microtubule associated protein Tau and also with Tubulin. Disruption of the Fyn-Tau interaction in cultured oligodendrocytes inhibits process outgrowth. Rafts may be a site of signaltransduction within the glial-axonal unit and serve to polarise vesicle traffic towards the glial axonal contact site.
The Sorting of Myelin Components in Oligodendrocytes
The generation of the myelin sheath involves the seIective targeting to and exclusion of distinct proteins and lipids to the specialised subdomains of myelin: e.g. compact myelin, the adaxonal membrane and the paranodes. We are using primary cultures of oligodendroglia to analyse the cell biological principles underlying this sorting out of cellular proteins and lipids to generate myelin. We have shown that oligodendrocytes use lipid rafts to sort the main myelin protein, PLP into the forming sheath.
|Fig. 1. Stages in the life of an oligodendrocyte|
Future Projects and Goals
1) Elucidation of the adhesion molecules regulating the migration of glial progenitor cells and their connection to the cell cytoskeleton. In particular, the role of the AN2/NG2 proteoglycan in this process.
2) What is the role of NG2+ glia at synapses: do neurons express receptors for the glycoprotein?
3) What is the role of the antibody against AN2 in the pathology of MS? Are specific regions of the molecule immunogenic in patients?
4) Analysis of the axon-glial interaction and signaltransduction: Definition of the signals required to initiate and maintain myelination. The role of the src kinase members fyn and lyn expressed by glia in this process. What are the axonal and glial partner adhesion molecules? What is the role of raft-associated cytoskeleton?
5) Definition of the cellular sorting and transport processes utilised by oligodendrocytes to build up the domain structure of myelin. How and where in the cell are the different microdomains generated, leading ultimately to the subdomains of myelin? What is the role of neuronal contact in this process?
|Fig. 2. Migratory oligodendrocyte progenitor cells express the AN2 gylcoprotein; Oligodendrocytes express the myelin lipid sulfatide and the cytoskeletal protein Tau|