Deliverables N-syndecan as a novel receptor for GDNF Syndecan-3 is a heparan sulphate proteoglycan (HSPG) that is profoundly expressed during brain development and maturation. We have found that GDNF interacts directly with syndecan-3 endogenously expressed by rat C6 glioma cell line. Affinity labelling of C6 cell with I125-GDNF followed by chemical cross-link and immunoprecipitation with syndecan-3 antibodies revealed high molecular weight complex. The complex formation was strongly inhibited by cell treatment with heparinase, soluble heparin and HB-GAM. Surprisingly, C6 cells treatment with PI-PLC, the enzyme that cleaves GPI-anchor thus releasing conventional GDNF co-receptor GFR?1, results in no attenuation of GDNF interaction with syndecan-3. This suggests that GDNF binds directly to HSPG syndecan-3. Role of GDNF ligands and receptors in lesion-induced neurogenesis GDNF was delivered using intra-striatal infusion for one week after stroke. Animals were then given the mitosis marker BrdU and we analyzed cell proliferation in the subventricular zone. The GDNF infusion caused a significant (33%) increase of cell proliferation in the ipsilateral subventricular zone as compared to vehicle-injected animals. NCAM as a signaling receptor for GDNF family ligands We have identified the neural cell adhesion molecule NCAM as the p135 signaling receptor for GDNF family ligands. Association of NCAM with GFR?1 was found to downregulate NCAM-mediated cell adhesion and to promote high affinity binding of GDNF to p140NCAM, resulting in rapid activation of cytoplasmic protein tyrosine kinases Fyn and FAK in cells lacking RET. GDNF stimulated Schwann cell migration and axonal growth in hippocampal and cortical neurons via binding to NCAM and activation of Fyn, but independently of RET. These results uncover an unexpected intersection between short- and long-range mechanisms of intercellular communication and reveal a pathway for GDNF signaling which does not require the RET receptor. Overexpression of GDNF in the intact nigrostriatal dopamine system Overexpression of GDNF in the intact nigrostriatal dopamine system results in downregulation of tyrosine hydroxylase (TH), a rate limiting enzyme in dopamine synthesis. However, autoradiography binding to dopamine receptors (D1 and D2), and dopamine transporter (DAT) in the striatum, show no significant change indicating that the reduction in TH does not result in any marked decrease in dopamine levels. We also show that the loss of TH is not a result of loss of dopaminergic cells in the substantia nigra since there is a normal number of dopamine neurons as determined by markers independent of TH. This effect was not seen by applying Artemin, another GDNF family ligand in the same paradigm. ENS progenitors are co-ordinately controlled by the endothelin receptor EDNRB and the GDNF receptor RET Two signalling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We have demonstrated that interaction between these signalling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhanced the effect of RET signalling on the proliferation of uncommitted ENS progenitors. In addition, we found novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A was a key component of the molecular mechanisms that integrate signalling by the two receptors. These data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals. Formation of neuronal synapses by ligand-mediated cell adhesion The GDNF receptor GFR1 is enriched at pre and postsynaptic compartments in hippocampal neurons, suggesting that it could participate in synapse formation. GDNF triggered trans-homophilic binding between GFR?1 molecules, and cell adhesion between GFR?1-expressing cells. In the presence of GDNF, immobilized GFR?1 induced localized presynaptic differentiation in hippocampal and cortical neurons as visualized by clustering of vesicular presynaptic proteins and neurotransmitter transporters. Presynaptic differentiation induced by GDNF was markedly reduced in neurons lacking GFR?1, and hippocampal synapses in Gdnf mutant mice showed reduced incorporation of presynaptic proteins, suggesting a role for GDNF signaling in hippocampal synaptogenesis in vivo. We propose that GFR?1 functions as a LICAM to establish precise synaptic contacts and induce presynaptic differentiation. Structure of the GDNF receptor GFR1 and its GDNF binding surface We have solved the 1.8 Å crystal structure of the GFR1 domain 3 and discovered a new protein fold. It is an all-alpha five-helix bundle stabilized by five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFR?1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF binding surface. Generation of encapsulated cells expressing GDNF To generate cell lines secreting GDNF suitable for encapsulation, stable clones were established from ARPE-19 cells (immortalized retinal pigment epithelial cells) by transfection with two different GDNF expression constructs. More than 200 clones were screened and among those, six clones secreting 90-133 ngGDNF/105 cells/24h in serum supplemented growth medium were chosen for further evaluation. When culturing the clones in a serum-free medium optimized for epithelial cells, GDNF secretion was increased approximately 2 folds for most clones. For all clones, stable GDNF secretion appeared to be maintained over 4 months in culture (and more than 20 passages) under proliferative conditions. Furthermore, GDNF secretion was stable at least 6 weeks after reaching confluency and GDNF bioactivity of conditioned media from the clones has been verified using a cell-based assay. After encapsulation of the clones in a standard device for experimental implantation into rat brains, GDNF release levels between 1-15 ng/device/24h could be achieved. Such devices could be used to obtain mechanistic insights into the neuroprotective role in appropriate animal models. GDNF promotes tubulogenesis of GFR-1-expressing MDCK cells by Src-mediated phosphorylation of MET receptor tyrosine kinase We have identified the receptor tyrosine kinase Met the receptor for hepatocyte growth factor (HGF) as the p150 downstream signaling component for both RET-dependent and -independent GDNF signaling. We have found that GDNF can partially restore ureteric branching morphogenesis in RET-deficient mice with severe renal hypodysplasia. In MDCK cells expressing GFR?1 but no RET, GDNF stimulated branching but not chemotactic migration, whereas both branching and chemotaxis were promoted by GDNF in cells co-expressing RET and GFR?1, mimicking HGF/Met responses in wild-type MDCK cells. However, GDNF did not bind to Met, implicating the participation of an alternative GDNF binding subunit. Met activation was mediated by Src family kinases. The GDNF-induced branching of MDCK cells required Src activation, whereas the HGF-induced branching did not. These data reveal a mechanism for GDNF-induced branching morphogenesis that is not dependent on the RET receptor. GFR 1-mediated GDNF signalling promotes the differentiation and tangential migration of cortical GABAergic neurons GDNF signalling via GFR1 was found to promote the differentiation of ventral precursors into GABAergic cells, enhancing their neuronal morphology and motility. GDNF stimulated axonal growth in cortical GABAergic neurons, and acted as a potent chemoattractant for those cells in organotypic slice cultures. All the effects of GDNF observed required GFR1 but neither RET nor NCAM, the two transmembrane signalling receptors known for GDNF. Mutant mice lacking GDNF or GFR1, but neither RET nor NCAM, showed reduced numbers of GABAergic cells in the cerebral cortex and hippocampus. Thus, one of the normal functions of GDNF signalling via GFR1 in the developing brain is to support the differentiation and migration of cortical GABAergic neurons. The lack of involvement of RET or NCAM in these processes suggests the existence of additional transmembrane effectors for GDNF. Analysis of gene expression during DA neuron differentiation and after depolarization We have used expanded DA cultures to identify genes important in DA differentiation. Oligonucleotide microarray analysis of 15866 genes showed that the number of genes up- and downregulated after 30 and 120 of exposure to high potassium were 62(up)/117(down) and 191(up)/92(down), respectively. Many of these genes are involved in important physiological processes. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
