Connexin26 expression in brain parenchymal cells demonstrated by targeted connexin ablation in transgenic mice

Abstract

Astrocytes are known to express the gap junction forming proteins connexin30 (Cx30) and connexin43 (Cx43), but it has remained controversial whether these cells also express connexin26 (Cx26). To further investigate this issue, we examined immunofluorescence labelling of glial connexins in wild-type vs transgenic mice with targeted deletion of Cx26 in neuronal and glial cells (Cx26fl/fl:Nestin-Cre mice). The Cx26 antibodies utilized specifically recognized Cx26 and lacked cross reaction with highly homologous Cx30, as demonstrated by immunoblotting and immunofluorescence in Cx26-transfected and Cx30-transfected C6 glioma cells. Punctate immunolabelling of Cx26 with these antibodies was observed in leptomeninges and subcortical brain regions. This labelling was absent in subcortical areas of Cx26fl/fl:Nestin-Cre mice, but persisted in leptomeningeal tissues of these mice, thereby distinguishing localization of Cx26 between parenchymal vs non-parenchymal tissue. In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic protein (GFAP). Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes and its ultrastructural localization in individual gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes.

Loss of astrocyte connexins 43 and 30 does not significantly alter susceptibility or severity of acute experimental autoimmune encephalomyelitis in mice

“…Thus, the absence of significant effect of Cx43/30 gene deletion on EAE may simply reflect the fact that these proteins are physiologically downregulated during acute EAE. No evidence of altered Cx26 expression, which is expressed at low levels in astrocytes (Nagy et al, 2011), was detected in these mice (Lutz et al, 2009). …”

Synergy between Electrical Coupling and Membrane Properties Promotes Strong Synchronization of Neurons of the Mesencephalic Trigeminal Nucleus

“…Many of these antibodies have been previously characterized for specificity by comparison of immunofluorescence and/or immunoblotting results in wild type mice vs. mice with knockout of the various connexins. Such characterization has included antibodies against Cx26 (Nagy et al, 2011), Cx30 (Lynn et al, 2011), Cx30.3, Cx31, Cx31.1 (Zheng-Fischhofer et al, 2006, 2007a,b), Cx32 (Nagy et al, 2003), Cx36 (Li et al, 2004), Cx47 (Li et al, 2008b) and Cx57 (Ciolofan et al, 2007). Similar comparisons between wild type and connexin KO mice have confirmed the specificity of antibodies against Cx29, Cx37 and Cx40 (Nagy, unpublished observations).…”

Panglial Gap Junctional Communication is Essential for Maintenance of Myelin in the CNS

“…Gap junction channels are formed among astrocytes (A/A), among oligodendrocytes (O/ O), and between oligodendrocytes and astrocytes (O/A), which results in formation of panglial networks (Wallraff et al, 2006;Maglione et al, 2010;Wasseff and Scherer, 2011). Since Cx29 does not form functional gap junction channels in cell culture experiments and Cx26 expression is limited to few gray matter astrocytes, panglial networks are mainly formed by astrocytic Cx43 and Cx30 and oligodendrocytic Cx47 and Cx32 (Ahn et al, 2008;Giaume and Theis, 2010;Nagy et al, 2011).…”