| At the onset of this dissertation it was not clear why CMTX patients primarily presented with PNS deficits, even though Cx32 was also expressed abundantly in oligodendrocytes. It was also not obvious what connexins were expressed by glia and if myelination in the central (CNS) nervous system required gap junctional communication. Therefore, the purpose of this thesis was to determine the expression and relative distribution patterns of connexins in CNS and PNS glia and explore their function in the maintenance of myelin. We approached this using genetic and immunohistochemical approaches. To identify novel CNS connexins I performed RT-PCR using degenerate primers, and identified Cx29, a novel connexin specifically expressed in glial cells. I showed that Cx29 expression was regulated in concert with myelin-associated genes and that it was a product of both oligodendrocytes and Schwann cells. I produced and characterized antibodies against Cx29, which revealed it to be highly localized in Schwann cells to the juxtaparanode and inner mesaxon, strikingly coincident with Kv1.2 present in the axolemma. However, the antibody studies suggested that Cx29 was either expressed in a subpopulation of oligodendrocytes or displayed different intracellular distributions in different subsets. To resolve this issue, I generated a Cx29 knockout mouse line that incorporated a histochemical reporter, which revealed Cx29 expression in all oligodendrocytes. Surprisingly, no gross motor defects or obvious ultrastructural abnormalities of CNS and PNS myelin were observed in Cx29 KO, Cx29/Cx32 dKO nor Cx29/Cx47 dKO animals. To explore the relationships of the oligodendrocyte and astrocyte connexins, I investigated the relative distributions of the six glial connexins by employing double and triple immunoflourescence microscopy using semi-thin sections (<1 mum) of adult mouse spinal cord. I found that astrocytes form two distinct classes of gap junctions with each other; those comprised of Cx26 and those comprised of Cx43 and Cx30. In addition, astrocytes establish two classes of intercellular channels with oligodendrocytes, heterotypic Cx26-Cx32 channels and heterotypic Cx30/Cx43-Cx47 channels that may also be heteromeric. In contrast, Cx29 did not co-localize with any of the other five connexins and does not appear to be involved conventional gap junctional intercellular communication. |
