Functional heterotypic interactions between astrocyte and oligodendrocyte connexins

Abstract

Human genetic diseases and mouse knockouts illustrate that the maintenance of central nervous system myelin requires connexin expression by both astrocytes and oligodendrocytes. Because these cell types express nonoverlapping sets of connexins, the intercellular channels formed between them must be asymmetric with regard to connexin content, defined as heterotypic. Here, we show that oligodendrocyte Cx47 can form heterotypic channels with astrocyte Cx43 or Cx30 but not Cx26, whereas oligodendrocyte Cx32 can functionally interact with astrocyte Cx30 or Cx26 but not Cx43. Thus, as many as four types of intercellular channels could be formed between astrocytes and oligodendrocytes.

Fig. 1

Incubation of HeLa cells with unlabeled avidin reduces background for neurobiotin injections. A) This HeLa strain does not exhibit gap junctional coupling as the junction-permeant tracer neurobiotin was detected only in the injected cell. However, a fine granular cytoplasmic background, likely reflecting biotin-containing compounds in HeLa mitochondria, reduces the sensitivity of the assay. B) Three days after the addition of unlabeled avidin to the culture media, a dramatic decrease in background was observed. Camera exposure time was identical to A. Scale bar: 50 μm.

Fig. 2

All glial connexins can form functional homotypic channels in HeLa cells. Lentiviral transduction was used to generate lines expressing specific glial connexins. Immunostaining (A-E) reveals an orthodox distribution including puncta localized to apposing membranes of adjacent cells (Scale bar: 30 μm). HeLa cells expressing individual glial connexins were able to transfer neurobiotin (F-J), whereas an empty vector control was not (K). Scale bar: 50 μm.

Fig. 3

Oligodendrocyte Cx47 cannot form functional heterotypic channels with Cx32 or Cx26 but does so readily with Cx43 or Cx30. Cells expressing Cx47 (A-E, green) were plated with cells expressing Cx32 (A, blue), Cx26 (B, blue), an empty vector control (C, blue), Cx43 (D, blue) and Cx30 (E, blue). Neurobiotin injection revealed heterotypic transfer only for Cx47-Cx43 and Cx47-Cx30 pairs. Consistent with these data, no co-localization of Cx47 with either Cx32 (F) or Cx26 (G) was evident, whereas co-localization of Cx47 with Cx43 (H) or Cx30 (I) was robust (arrowheads). Scale bar: 50 μm.

Fig. 4

Oligodendrocyte Cx32 can form functional heterotypic channels with Cx30 or Cx26. Cells expressing Cx32 (A-D, green) were mixed with cells expressing Cx43 (A, blue), empty vector controls (B, blue), Cx30 (C, blue), or Cx26 (D, blue). Neurobiotin injection revealed heterotypic transfer only in Cx32-Cx30 and Cx32-Cx26 pairs (scale bar: 50 lm. Consistent with these data, Cx32 (E-G, red) did not co-localize with Cx43 (E, green) but did overlap (arrowheads) with Cx30 (F, green) and with Cx26 (G, green) when adjacent cells expressed those combinations of connexins. Scale bar: 50 μm.

Fig. 5

Primary astrocytes lacking Cx43 are communication negative. A) Primary astrocyte cultures were 95% GFAP-positive. B) RTPCR screen of control astrocytes reveals abundant Cx43 while astrocytes derived from a conditional Cx43KO showed very weak expression of Cx43, likely reflecting contamination of the culture with non-astrocytic cells. Both WT and KO cells express very low levels of Cx26 signal (< one copy per cell on average). No other connexin transcript was consistently observed (data not shown). C) Control astrocytes displayed multiple Cx43-positive puncta reminiscent of gap junctions. D) Most KO astrocytes lacked any detectable signal. E) Control astrocytes displayed extensive transfer of neurobiotin. F) Cx43KO astrocytes exhibited no transfer.

Fig. 6

The pattern of heterotypic communication in primary astrocytes is identical to HeLa cells. Lentiviral transduction of astrocytes was at least as efficient as in HeLa cells allowing experiments to be completed well before (≤15 DIV) the appearance of endogenous Cx30 (21-28 DIV). As with HeLa cells, neurobiotin (NB) transfer was not observed in pairings of Cx47-Cx32 (A), Cx47-Cx26 (B), or Cx32-Cx43 (C) but was readily detected in pairings of Cx47-Cx43 (D), Cx47-Cx30 (E), Cx32-Cx30 (F), and Cx32-Cx26 (G). Scale bar: 50 μm.