Transgenic expression of human connexin32 in myelinating Schwann cells prevents demyelination in connexin32-null mice

Abstract

Mutations in Gap Junction beta1 (GJB1), the gene encoding the gap junction protein connexin32 (Cx32), cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X), an inherited demyelinating neuropathy. We investigated the possibility that the expression of mutant Cx32 in other cells besides myelinating Schwann cells contributes to the development of demyelination. Human Cx32 was expressed in transgenic mice using a rat myelin protein zero (Mpz) promoter, which is exclusively expressed by myelinating Schwann cells. Male mice expressing the human transgene were crossed with female Gjb1/cx32-null mice; the resulting male offspring were all cx32-null (on the X chromosome), and one-half were transgene positive. In these transgenic mice, all of the Cx32 was derived from the expression of the transgene and was found in the sciatic nerve but not in the spinal cord or the liver. Furthermore, the Cx32 protein was properly localized (within incisures and paranodes) in myelinating Schwann cells. Finally, the expression of human Cx32 protein "rescued" the phenotype of cx32-null mice, because the transgenic mice have significantly fewer demyelinated or remyelinated axons than their nontransgenic littermates. These results indicate that the loss of Schwann-cell-autonomous expression of Cx32 is sufficient to account for demyelination in CMT1X.

Figure 1.

A rat Mpz promoter/GJB1 transgene cassette is expressed in peripheral nerve. A, The structure of the human GJB1/Cx32 gene. In myelinating Schwann cells, Cx32 transcripts are initiated from the P2 promoter; in the liver, transcripts are initiated from the P1 promoter (Neuhaus et al., 1995, 1996; Söhl et al., 1996). B, The structure of the transgene. The 1.1 kB rat Mpz/P0 promoter is joined upstream of exon 1b of the human GJB1/Cx32 gene. The positions of the primer pairs used to amplify cDNA, <1> and <4> and <P0> and <2>, are indicated. C, Analysis of endogenous versus transgene expression by semiquantitative RT-PCR. The top panel shows human/mouse Cx32/cx32 cDNA amplified with primers (<1> and <4>) that hybridize with a sequence that is identical in human and mouse Cx32/cx32. The undigested (U) PCR product is 553 bp; HhaI (H) cuts mouse cx32 but not human Cx32, MscI (M) cuts human Cx32 but not mouse cx32; the double digestion (D) proves that no full-length product remains. The bottom panel shows similarly digested RT-PCR products from adult transgenic sciatic nerve. Densitometric quantitation of the mouse- and human-specific bands in the MscI-cut lane indicates that the ratio of transgene/human Cx32 mRNA are ∼1 and 3 for lines 90 and 96, respectively. MK, DNA size markers.

Figure 2.

Expression of Cx32 and P₀ in peripheral nerve and liver. A, Immunoblot analysis of Cx32. Protein extracts of sciatic nerve and liver from P445 TG⁺ (lanes 1 and 2) and TG^- (lanes 3 and 4) cx32^-/Y littermates and a P400 male wild-type mouse (lanes 5 and 6) were probed with a rabbit antiserum (Zymed) against the Cx32 intracellular loop (exposure time, 2 min). The single and double arrowheads indicate Cx32 monomers and dimers, respectively. Note that Cx32 detected in the sciatic nerve but not in the liver of TG⁺ mice (compare lanes 1 and 2), that there is relatively more Cx32 in TG⁺ mice than in wild-type mice (compare lanes 1 and 5), and that some Cx32 appears to be partially degraded (lane 1, arrow). Any bands that are found in (TG^-) cx32-null liver and nerve are nonspecific, such as the prominent bands in liver (asterisks). B, Reprobing the same blot with a rabbit antiserum against P₀ demonstrates comparable levels of P₀ (arrow) in all nerves (exposure time, 20 s). C, Coomassie-stained gel after transfer shows loading of sciatic nerve and liver samples. The position of P₀ is indicated (arrow).

Figure 3.

Myelinating Schwann cells but not oligodendrocytes express a Mpz-Cx32 transgene. These are confocal images of unfixed s.c. and attached vr from a P400 wild-type mouse (A) and P385 TG^- (B) and TG⁺ (C) cx32^-/Y littermates. The sections were labeled with a rabbit antiserum against the cytoplasmic loop of Cx32 (Chemicon), a mouse monoclonal antibody against MOG, and a rat monoclonal antibody against NF-H. The top panels depict Cx32 staining alone; the bottom panels show merged images of Cx32 (green), MOG (red), and NF-H (blue). Note that MOG is localized to the CNS myelin sheaths in the ventral funiculus; that NF-H is found in both CNS and PNS axons; and that Cx32 is present in both the roots and the cord of wild-type mice (A), in neither the roots nor the cord of TG^- mice (B), and in the roots but not in the cord of TG⁺ mice (C). The exposure to visualize Cx32 immunoreactivity for B was as long as that for C. Scale bar, 20 μm.

Figure 4.

Human Cx32 localizes to noncompact myelin. These are confocal images of unfixed teased sciatic nerve fibers from a P400 wild-type mouse (A) and P385 TG^- (B) and TG⁺ (C) cx32^-/Y littermates. The fibers were immunostained with a mouse monoclonal antibody against Cx32, a rabbit antiserum against E-cadherin, and a rat monoclonal antibody against NF-H, as indicated. Apposed arrowheads mark nodes, which are flanked by Cx32-positive paranodes in wild-type and TG⁺ mice. Prominent E-cadherin staining is seen in incisures (arrowheads) and outer mesaxons (arrows). The large aggregates of Cx32 in the outer mesaxons (circled), incisures, and paranodes of cx32^-/Y TG⁺ fibers (C) are not seen in wild-type fibers (A). Scale bars, 10 μm.

Figure 5.

Transgenic expression of human Cx32 prevents demyelination. These are images of semithin sections of femoral motor nerve from TG⁺ and TG^- mice at P158, P250, and P365 cx32^-/Y littermates. The TG⁺ nerves look normal at P158 and P250, but there are 30 de/remyelinated axons in the P365 nerve. The number of de/remyelinated axons at P158, P250, and P365 in TG^- nerves was higher at every age (135, 166, and 297, respectively). Arrowheads mark the perineurium. The areas outlined by the rectangles are enlarged in Figure 6, A and B. Scale bar, 20 μm.

Figure 6.

Transgenic expression of human Cx32 prevents demyelination. These are photomicrographs of semithin sections of the femoral motor branch (A, B; P250) and lumbar ventral roots (C, D, P250; E, F, P365) of TG^- and TG⁺ cx32^-/Y mice. Note that the myelinated axons appear normal in all TG⁺ samples. Demyelinated axons are indicated (^*) in the TG^- samples, and remyelinated axons (r) as well as disrupted/split myelin sheaths (#) and macrophages containing myelin debris (m) are labeled in the P250 femoral motor branch. Scale bar, 10 μm.

Figure 7.

Transgenic expression of human Cx32 prevents segmental demyelination. These are differential interference contrast images of teased fibers from osmicated femoral motor branches of TG^- or TG⁺ P387 cx32^-/Y littermates. In each panel, three teased fibers are labeled (1-3); apposed arrowheads mark the nodes of normal-appearing myelinated axons. Note the one normal-appearing myelinated axon (1) in the TG^- root, along with two axons with abnormally thin myelin sheaths (2 and 3) that are associated with supernumerary Schwann cell nuclei (n) and their cellular processes (arrows). All three TG⁺ teased fibers appear normal; the axon (a) and incisures (arrowheads) are clearly seen in one fiber in the correct focal plane (3). Scale bar, 10 μm.

Figure 8.

The proportion of abnormally myelinated axons in TG^- or TG⁺ femoral motor nerves. This plot shows the proportion of abnormally myelinated axons in the femoral motor nerves at P158, P250, and P365. The means are shown at each age, separately for TG^- and TG⁺ animals.