Connexin29 is uniquely distributed within myelinating glial cells of the central and peripheral nervous systems

Abstract

Although both Schwann cells and oligodendrocytes express connexin32 (Cx32), the loss of this connexin causes demyelination only in the PNS. To determine whether oligodendrocytes might express another connexin that can function in place of Cx32, we searched for novel CNS-specific connexins using reverse transcriptase-PCR and degenerate primers. We identified Cx29, whose transcript was restricted to brain, spinal cord, and sciatic nerve. Developmental expression of Cx29 mRNA in the CNS paralleled that of other myelin-related mRNAs, including Cx32. In the CNS, Cx29 antibodies labeled the internodal and juxtaparanodal regions of small myelin sheaths, whereas Cx32 staining was restricted to large myelinated fibers. In the PNS, Cx29 expression preceded that of Cx32 and declined to lower levels than Cx32 in adulthood. In adult sciatic nerve, Cx29 was primarily localized to the innermost aspects of the myelin sheath, the paranode, the juxtaparanode, and the inner mesaxon. Cx29 displayed a striking coincidence with Kv1.2 K(+) channels, which are localized in the axonal membrane. Both Cx29 and Cx32 were found in the incisures. Cx29 expressed in N2A cells did not induce intercellular conductances but did participate in the formation of active channels when coexpressed with Cx32. Together, these data show that Cx29 and Cx32 are expressed by myelinating glial cells with distinct distributions.

Fig. 1.

Cx29 is among the most divergent connexins.A, Alignment of Cx29 and hCx31.3 reveals 61% amino acid identity. Nonidentical residues are boxed.B, Dendrogram illustrating phylogenetic relationships based on CLUSTAL algorithm. These data suggest that Cx29 and hCx31.3 are orthologs and are among the most divergent members of the connexin family.

Fig. 2.

Cx29 is a nervous system-specific connexin and is expressed coordinately with other myelin-related genes. Northern blots of total RNA were hybridized with the indicated cDNA probes.A, Cx29 transcript is detected only in brain, spinal cord, and sciatic nerve of adult mice. B, C, Developmental pattern of Cx29 mRNA expression in mouse cerebellum (B) and cerebrum (C) generally parallels that of PLP (only the largest PLP transcript is shown). However, Cx29 is expressed earlier in the cerebellum. D, E, Male MD rats have undetectable levels of Cx29 and Cx32 mRNA compared with their age-matched unaffected male littermates.F, Cx29 is expressed before Cx32 mRNA in developing rat sciatic nerves and is barely detectable in adult nerves. G, H, The level of Cx29 mRNA increases in the distal stumps of crushed/regenerating adult rat sciatic nerves, paralleling those of P₀ and Cx32, but in transected/permanently denervated distal stumps, Cx29, P₀, and Cx32 mRNAs do not increase. NGFR, Nerve growth factor receptor.

Fig. 3.

Cx29 is expressed by small cells in both white and gray matter of spinal cord. In situ hybridization in transverse sections of adult mouse spinal cord. A, An antisense Cx29 RNA probe corresponding to the coding region stains small cells throughout the cord.B, A sense Cx29 probe exhibits relatively low background. C, The pattern of hybridization with PLP/DM20 probe is similar to Cx29, but a larger number of cells are stained, especially in the white matter.

Fig. 4.

Cx29 is not expressed by neurons or astrocytes in spinal cord. In situ hybridization for Cx29 in transverse sections of spinal cord combined with immunocytochemistry for cell-specific markers is shown. A, B, Cells immunolabeled for NeuN do not correspond to cells labeled byinsitu hybridization for Cx29 mRNA (arrows, examples of NeuN-positive cells;arrowheads, Cx29-positive cells). C, Similarly, GFAP-positive cells do not express Cx29 mRNA (arrows, examples of GFAP-positive cells;arrowheads, Cx29-positive cells).

Fig. 5.

Cx29 displays anomalous behavior in Western blots. Affinity-purified anti-Cx29 antibody was produced against a GST fusion protein containing C-terminal residues 220–258. Lane 1, Ten nanograms of purified GST fusion protein. Lane 2, COS cells transfected with rCx32 provide negative control for anti-Cx29 antibody. Lane 3, COS cells cotransfected with rCx32 and Cx29. Bands at ∼29, 60, and 85 kDa were detected. Lane 4, Total proteins from adult mouse brain. Strong labeling of several bands in addition to the ones detected in COS cells was observed, possibly resulting from proteolytic digestion (see Discussion). Lane 5, Total proteins from mouse spinal cord present a similar pattern to brain. Lane 6, Total proteins from mouse sciatic nerve. In sciatic nerve, the 60 kDa band was more abundant relative to the 29 kDa band.

Fig. 6.

Cx29 and Cx32 are expressed in different subsets of spinal cord oligodendrocytes. Confocal images of horizontal sections of mouse spinal cord double labeled for Cx29 (A, B;red) and Cx32 (A; green) or Caspr (B; green) are shown. Cx29 labels the internodes and juxtaparanodes (A, B;light blue arrowheads) of small diameter fibers and a few somata (A; yellow arrowheads). The Cx32 antibody labels the somata of intrafascicular oligodendrocytes (A; white arrowheads), and labeling extends into fiber tracts. The Caspr antibody (B) strongly labels the paranodal axolemma, thereby illuminating the point that large axons have little Cx29 immunostaining (B;dark blue arrowheads).

Fig. 7.

Cx29 and Cx32 are distinctly localized in myelinating Schwann cells. These are images of unfixed teased fibers from rat sciatic nerve, after double labeling for Cx29 (red) and Cx32 (green). Cx32 is strongly expressed in the outermost aspects of paranodes (white arrowheads) and incisures (white arrows). Cx29 is localized primarily to the innermost aspects of paranodes and juxtaparanodes (yellow arrowheads) and more uniformly in incisures (white arrows). Cx29 was enriched in the inner mesaxon (bracketed by blue arrowheads), which spans the internodal region.

Fig. 8.

Cx29 is localized to the inner mesaxon. These are images of fixed teased fiber from rat sciatic nerve, after double labeling for Cx29 (red) and either MAG (A; green) or Kv1.2 (B;green). A, The internodal Cx29 signal consists of a pair of lines (bracketed by red arrowheads) that flank a single line of MAG staining (bracketed by green arrowheads); Cx29 and MAG colabel incisures.B, Cx29 and Kv1.2 are strikingly aligned both at the inner mesaxon (bracketed by bluearrowheads) and at the innermost aspect of incisures (arrows). The double nature of the Cx29 and Kv1.2 signals is not apparent at this magnification. Cx29 immunoreactivity is present in the paranode (red region nearest node of Ranvier) and juxtaparanode (yellow area marked bywhite arrowheads).

Fig. 9.

Immuno-EM of Cx29. These are images of teased fibers that were fixed in 0.5% glutaraldehyde, immunostained for Cx29 using DAB as the substrate for peroxidase, and processed for EM. Two teased fibers are illustrated in A, unstained semithin sections are shown in B and C, and thin sections photographed with an electron microscope are shown inD and E. In all panels, there is DAB throughout incisures (arrowheads) and in the innermost aspects of the juxtaparanodal/paranodal regions (arrows) of myelinating Schwann cells.SC, Schwann cell nucleus; M, myelin sheath; A, axon. Double arrowheads, Node of Ranvier. Scale bars: A–C, 10 μm; D, E, 1 μm.

Fig. 10.

Developmental expression of Cx29. These are images of sections of rat sciatic nerve, double labeled for Cx29 (red; A, C) and MAG (green; B, D). At P3, MAG (B) is mostly diffusely distributed throughout the internodal region, whereas Cx29 (A) is less diffusely localized and more concentrated adjacent to developing nodes. By P10, both MAG and Cx29 display similar distributions (C, D).

Fig. 11.

Cx29 forms heteromeric channels with Cx32. Connexins were expressed in N2A cells by transient transfection along with fluorescent markers, and dual whole-cell patch recording was performed. A, Normalized conductance–transjunctional voltage (G_j–V) relationship in control pairs expressing only Cx32. Junctional conductance is symmetrical and displaysV_j-dependent inactivation (n = 7). TheG_j–V_jrelationship fitted a double Boltzmann distribution whose fit is superimposed (solid line). B,G_j–V relationship in pairs expressing Cx32 in one cell and both Cx32 and Cx29 in the other (n = 7). The relationship is no longer symmetrical. The V_j indicated on thex-axis is applied to the cell containing both connexins.C–H, Representative examples of recordings from one cell pair with a single heteromeric channel. A transjunction potential of 100 mV was imposed. The Cx32-only cell (top traces) exhibits an ∼70 pS channel that rapidly transitions to several substates. The heteromeric channel exhibits similar single-channel conductances, but open states are prolonged without the frequent transitions to substates. The difference in gating behavior is exemplified in I, displaying the average of 30 of such sweeps. The average peak current for both polarities is 7–8 pA, but the time-dependent current decay was well fitted by a single exponential function (solid line) with a larger time constant for the heteromeric than for the homomeric side.

Fig. 12.

The organization of connexins in CNS and PNS myelin sheaths. This is a schematic depiction of a myelinated fiber that has both a CNS and PNS myelin sheath. The myelin sheath and axon are depicted as hemisected, and the internodal, paranodal, juxtaparanodal, and nodal regions are shown. In the PNS, Cx29 is depicted as localized to the adaxonal Schwann cell membrane (in the juxtaparanodal region, at the inner mesaxon, and at the innermost aspect of incisures) and between the glial loops in the paranodal region. In the CNS, Cx29 is depicted as localized to the adaxonal oligodendrocyte. Cx32 is localized to the incisures and paranodes of myelinating Schwann cells and the outer membrane of large myelinated fibers in the CNS [modified from Arroyo and Scherer (2000) and used with permission of Springer-Verlag].