Exploration of culture conditions that support the growth of human oligodendrocyte progenitors and examination of neuronal expression of a unique G-protein, Galphaz

Abstract

The work presented in this thesis addresses two questions relevant to central nervous system function. The first explores culture conditions that could lead to the expansion in vitro of human oligodendrocyte (OL) precursor cell populations. The second addresses the potential role of Galphaz in neuronal development and in differentiated neurons.;Using 2nd trimester human frontal cortical tissues results show that the application of rat OL isolation procedures and the growth and/or differentiation factors, PDGF, bFGF, PDGF+bFGF and TGFbeta1, did not replicate the same results in human tissue. We show, however, that a population of PSA-NCAM+ putative multipotential stem cells increases from approximately 5 to 11 to 16% of the culture over time. Furthermore, we also show that the methodologies established in the rat result in the isolation and maintenance of human neurons. Neurons maintained under these conditions undergo morphological differentiation, express neuron-specific epitopes and voltage- and ligand-gated ion channels.;In the second part of this thesis, studies were initiated leading to the analysis of the role of Galphaz during nervous system development and in differentiated neurons. Results demonstrate that Galphaz protein exhibits an increasing pattern of expression in the rat neocortex during target innervation, neuritogenesis and synaptogenesis. Parallel studies using human neocortical extracts suggest a similar phenomenon occurs in the human. Studies are confirmed using culture models of both rat and human origin. Immunocytochemical analyses of developing neurons in vitro reveals that Galphaz is located throughout the cell soma and developing neuritic processes during all stages of development.;Somatic Galphaz, is present within, but is not confined to, the endoplasmic reticulum in differentiated neurons Additional intracellular sites of activity may be separate from those occupied by other Galphai/o family members as results also show that Galphaz localizes to discrete sites apart from other Galphai/o family members. Unique sites of activation do not include synapses but may microtubules (MTs), as a subpopulation of MT-associated Galphaz is found in an active conformation.;An interesting possibility stemming from this dissertation is that Galphaz participates in a number of signal transduction pathways by shuttling between cellular domains in a persistently activated state.