Connexin expression, connexin-mediated communication, and the effect of connexin overexpression in an in vitro model of human neuronal differentiation.

Abstract

The connexin (Cx) family of transmembrane proteins oligomerize to form hemichannels, permitting communication with the extracellular environment, and gap junctions, forming the basis for intercellular communication between adjacent cells. Although hemichannel communication remains unexplored, it is well established that gap junction intercellular communication is maximal in the developing central nervous system prior to formation of functional chemical synapses. The NT2/D1 (NT2) in vitro system, which can be terminally differentiated to hNT cultures containing human central nervous system neurons, was used as a starting point to further our understanding of the functional significance of Cx proteins and Cx-mediated communication in influencing neural progenitor fate. In Chapter 1, several Cxs, typically expressed by the mammalian central nervous system, were analyzed for expression in NT2 and hNT cultures. Cx30, Cx36, Cx37, and Cx43 were detected in undifferentiated NT2 cultures. Cx36, Cx43, and Cx46.6 were detected in all hNT cultures tested while Cx30 and Cx37 were detected in only a subset of hNT cultures. In Chapter 3, fluorescent dye transfer assays validated previous reports of decreased gap junction intercellular communication in hNT cultures compared to NT2 cultures. A series of dye uptake assays revealed inducible hemichannel activity in NT2s but not hNTs suggesting that like gap junction intercellular communication, hemichannel activity decreases following terminal neuronal differentiation. This marks the first report of hemichannel activity in neural progenitors. In Chapter 4, the specific role of Cx32 in neuronal differentiation was addressed through transfection of NT2s with Cx32. Cx32 expression blocked terminal neuronal differentiation without affecting oligodendrocyte differentiation as determined by lineage analysis of NT2s and hNTs. We propose a model where Cx32 expression by neural progenitors prevents differentiation to neurons and promotes differentiation to oligodendrocytes in NT2 cell through activation of hemichannels.