Use of PC12 cells to characterize PAFR-GPCR-mediated activity in neural precursors.

Abstract

Platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator produced by neurons and glial cells. In adult central nervous system (CNS), physiological concentrations of PAF have been shown to mediate long-term potentiation, the purported cellular basis of mammalian learning and memory. Elevated levels of PAF are implicated in the pathophysiology of a number of neurodegenerative diseases, ischemia-reperfusion injury, human immunodeficiency virus-1 (HIV-1) associated dementia, and the developmental brain disorder Miller-Dieker Syndrome (MDS). Three neuropathologies are associated with sustained PAF exposure. It is not clear how these PAF-mediated effects are initiated in CNS. A seven transmembrane G-protein coupled receptor (PAFR-GPCR) has been cloned and mRNA is expressed in CNS. In addition, distinct intracellular receptor isoforms (iPAFRs) in rodent cortical extracts have been characterized pharmacologically. It is not known which of these PAF binding proteins initiate PAF-mediated neuropathology. Our ability to detect PAF receptors is further hindered by a lack of commercial PAF antibodies. To address these issues, in this thesis, I sought to: (1) Identify a neural precursor cell line capable of differentiating between PAFR-GPCR and PPAFR-initiated biological activity, (2) Determine the effect of PAFR-GPCR activation on differentiation of neural precursors to a neuronal phenotype, and (3) Characterize a new PAFR-GPCR antibody to facilitate localization of protein in CNS and CNS cell lines. (Abstract shortened by UMI.)