3 0

3 0.001) showing that Cx43/Cx32 deficiency results in a striking loss of myelinating cells in the cortex of 60-d-old mice. Generation and characterization of new polyclonal Cx47 antibodies To quantify the amount of Cx47 protein in the different transgenic connexin mouse mutants, we had to generate new Cx47 antibodies because commercially available Cx47 antibodies were found to give a false-positive Eprodisate signal at 47 kDa in immunoblot analyses of Cx47KO tissues. astrocytic Cx43 protein and that its gap junctional channel function is not necessary for Cx47 stabilization. In consequence, Cx43/Cx32dKO mice additionally lack Cx47 expression and therefore cannot form oligodendrocytic gap junctions, which explains the phenotypic similarities to Cx32/Cx47dKO mice. Our findings provide strong evidence that phosphorylation and stability of oligodendrocytic Cx47 proteins is dependent on astrocytic Cx43 expression. These results further unravel the complexity of panglial networks and show that results of previous studies using astrocytic Cx43-deficient mice have to be reconsidered. Introduction Complex multicellular organisms are dependent on direct cellCcell communication through gap junction (GJ) channels. GJ channels are characterized by their ability to allow diffusion of ions and metabolites ( 1200 Da) (Bruzzone et al., 1996). They are composed of two connexons (hemichannels), whereby each connexon is provided by one adjacent cell. Each connexon is composed of six connexin protein subunits. Identical connexons form homotypic channels, whereas heterotypic channels are composed of two different hemichannels. Neurons and macroglial cells express distinct sets of connexin isoforms. Astrocytes express Connexin 30 (Cx30) and Cx43. In addition, Cx26 is expressed in a small subpopulation of gray matter astrocytes (Nagy et al., 2011). Oligodendrocytes express Cx47, Cx32, and Cx29, but Cx29 does not form functional GJ channels (Kleopa et al., 2004). Expression of oligodendrocytic and astrocytic connexins results in functional intra-astroglial (A:A), intraoligodendroglial (O:O), and interastro-oligodendroglial (A:O) coupling (Wallraff et al., 2006; Maglione et al., 2010; Wasseff and Scherer, 2011). Several connexin single and double knock-out mice (dKO) have been investigated to understand the function of glial connexins participating in panglial networks. Single KOs did not result in gross morphological alterations (Sutor et al., 2000; Odermatt et al., 2003; Teubner et al., 2003; Theis et al., 2003; Eiberger et al., 2006; Nagy et al., 2011). Loss of both astrocytic connexins (Cx30 and Cx43) leads to dismyelination and vacuolization of gray as well as white matter regions (Lutz et al., 2009), whereas mice deficient for both oligodendrocytic connexins, Cx32 and Cx47, show severe myelin abnormalities and die at approximately postnatal day 42 (P42) (Menichella et al., 2003; Odermatt et al., 2003). In addition, mice deficient for one astrocytic (Cx30) and one oligodendrocytic (Cx47) connexin show complete loss of A:O coupling, early onset myelin pathology, motor impairments, and 40% of Cx30/Cx47dKO animals die between P42 and P90 (Tress et al., 2012). Altogether, these studies indicate that A:O coupling is essential for myelin maintenance and are in line with a theoretical model of the coupling situation in the CNS Eprodisate based on cell culture experiments, in which A:O coupling can be established by Cx30:Cx32, Cx30:Cx47, and Cx43:Cx47 heterotypic channels, whereas combinations of Cx43 and Tmem17 Cx32 cannot form functional channels (Orthmann-Murphy et al., 2007; Magnotti et al., 2011a). Recently, a study on Cx43 and Cx32 double-deficient mice revealed severe phenotypic abnormalities with astrocyte loss, white matter vacuolization, and early death at 16 weeks after birth (Magnotti et al., 2011b). This was surprising because Cx30:Cx47 channels should maintain Eprodisate functional coupling among astrocytes and oligodendrocytes in Cx43/Cx32dKO mice, whereas O:O and A:A coupling should be maintained by Cx47 and Cx30 expression, respectively. With the present study, we aimed to identify the mechanism underlying the unexpected phenotype of Cx43/Cx32dKO mice and provide new insights into the function and contribution of distinct oligodendrocytic and astrocytic connexins. We show that Cx47 is phosphorylated and that Cx47 expression in oligodendrocytes and phosphorylation of Cx47 depend on astrocytic Cx43 expression in the mouse brain. Therefore, the phenotype of Cx43/Cx32dKO mice Eprodisate results from loss of both oligodendrocytic connexins Cx32 and Cx47. Materials and Methods Animals..