No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

An abundant population of glial cells in the central nervous system (CNS) is identified by the specific expression of the NG2 chondroitin sulphate proteoglycan (CSPG) and are termed NG. NG2-slia are phenotypically distinct from astrocytes, oligodendrocytes, ependyma and microglia, and represent a fifth major type of glia in the brain. Generally, NG2-glia have been considered to be oligodendrocyte progenitor cells (OPCs), but there is evidence that NG2-glia possess neural stem cell-like properties, being able to generate oligodendrocytes, astrocytes and neurons. In addition, NG2-glia respond to different types of brain injury by a rapid and extensive proliferation, or gliosis, and contribute to the axon growth inhibitory glial scar and regeneration of oligodendrocytes. A notable feature of NG2-glia is that they have complex multibranched process fields that form multiple glutamatergic synaptic contacts with neurons. So far, the function of NG2-glial glutamatergic 'synapses' is unresolved. The overall aim of this thesis was to test the hypothesis that NG2-glia may be important for the stabilization and integrity of synapses, and that glutamate released at these synapses may regulate the proliferation and differentiation capacity of NG2-glia.

GPCRs are rapidly phosphorylated in response to stimulation and this regulates receptor function and signalling. This process is widely considered to be mediated by the GRK family of protein kinases. In the current thesis we extend this paradigm by examining the role of the casein kinases in the phosphorylation of the M3-muscarinic receptor. Investigation of a phosphorylation deficient mutant of the M 3-receptor (mutant 6), where 17 putative serine phospho-acceptor sites had been substituted, demonstrated that receptor internalisation and efficient coupling to the phospholipase C pathway are dependent on receptor phosphorylation. However, this receptor mutant coupled normally to the ERK and JNK kinase pathway and mediated calcium entry similar to the wild type receptor. The importance of the specific sites of phosphorylation in driving defined receptor processes was illustrated by the use of another phosphorylation deficient mutant of the M3-receptor where amino acids between Lys 370-Ser425 inclusive were removed. Specific reduction of CK1alpha and CK2 protein expression levels with siRNA resulted in decreased agonist-induced receptor phosphorylation and had no effect on receptor internalisation, indicating that phosphorylation by kinase/s other than the casein kinases (probably the GRKs) are required for internalisation. Following inhibition of CK2-mediated receptor phosphorylation, receptor stimulated Ins(1,4,5)P3 production and ERK activation occurred as normal. In contrast, siRNAs against CK1alpha reduced the ability of the receptor to stimulate Ins(1,4,5)P3 production whereas coupling to the ERK pathway was unaffected. The data presented here support the hypothesis that phosphorylation at specific sites is mediated by a range of receptor kinases that include CK1alpha, CK2 and the GRKs. It appears that site specific phosphorylation by particular kinases regulates defined receptor processes.

Human SH-SY5Y neuroblastoma cells were used to investigate mechanisms involved in phosphorylation of the transcription factor CREB (cAMP response-element binding-protein) after activation of two endogenously expressed Gq/11 -protein-coupled receptors, the M3 muscarinic acetylcholine (mACh) B2 bradykinin receptors. Stimulation with either methacholine (MCh) or bradykinin resulted in maximal increases in CREB phosphorylation within 1 min, with either a rapid subsequent decrease (bradykinin) to basal levels, or a maintained plateau response (MCh). Inhibitor studies were performed to assess the involvement of a number of potential kinases in signalling to CREB phosphorylation. Removal of extracellular Ca2+, inhibition of Ca2+/calmodulin-dependent protein kinases (CaMKs) and down-regulation of protein kinase C (PKC), resulted in reduced CREB phosphorylation after M3 mACh and B2 bradykinin receptor activation. Inhibition of MEK1/2 (MAPK/ERK kinase) by U0126 resulted in significantly reduced CREB phosphorylation levels after B2 bradykinin but not M3 mACh receptor activation. It has also been demonstrated that maintained phosphorylation of CREB is necessary for cAMP response element (CRE)-dependent gene transcription as the M3 mACh, but not the B2 bradykinin receptor activates both a recombinant CRE-dependent reporter gene, and endogenous c-Fos gene transcription.;Activation of the M3 mACh receptor is capable of increasing the processing of the amyloid precursor protein (APP) via an alpha-secretase pathway to yield the non-amyloidogenic secreted protein, sAPPalpha. This increase in sAPPalpha was shown to be dependent upon influx of Ca 2+ from the extracellular milieu. Inhibition of CaMKs and PKC resulted in reduced secretion of sAPPalpha, demonstrating a role for these kinases in M3 mACh receptor regulation of APP processing.

The regulation of mitogen-activated protein kinase (MAPK) activities by the group I metabotropic glutamate (mGlu) receptors, mGlu1a and mGlu5a, has been studied in Chinese hamster ovary (CHO) cells, where receptor expression is under inducible control. Both mGlu receptors stimulated comparable, robust and transient increases in the activity of the extracellular signal-regulated kinase (ERK) subgroup. Further, the mGlu1a, but not the mGlu5a receptor was found to mediate an increase in the activity of c-Jun N-terminal kinase (JNK). Examination of the signalling profile of mGlu1/mGlu5a receptor-mediated ERK activation revealed clear differences in the G-protein subpopulations involved, with only mGlu1a receptor-mediated ERK responses attenuated by pertussis toxin (PTx) pre-treatment. Both mGlu1a and mGlu5a receptor-mediated ERK activation occurred via mechanisms dependent on the non-receptor tyrosine kinase, Src, but independent of phosphoinositide 3-kinase activity, PKC and intracellular and/or extracellular Ca2+ concentration. Data also demonstrate a requirement for PDGF receptor tyrosine kinase activity in ERK activation by the mGlu1a, but not the mGlu4a receptor. The mGlu1a receptor-mediated JNK response, unlike ERK activation by the same receptor, was insensitive to PTx pre-treatment and occurred via mechanisms independent of intracellular and/or extracellular Ca2+ concentration, Src kinase and was unaffected by PKC down-regulation. The delayed onset of JNK activation by the mGlu1a receptor was not found to be a result of earlier ERK activation. Stimulations of mGlu1a/mGlu5a receptors, did not alter cellular proliferation, as measured by DNA synthesis, or have a marked effect on cytoskeletal organisation, as measured by immunocytochemistry, indicating that the activation of mitogenic signalling by these two mGlu receptors does not result in changes in growth in these cells. These studies highlight important differences in the activation and signalling pathways utilised to regulate MAP kinases.