Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin 32

Abstract

Mutations affecting the connexin 32 (Cx32) gene are associated with the X-linked form of the hereditary peripheral neuropathy Charcot-Marie-Tooth disease (CMTX). We show that Cx32-deficient mice develop a late-onset progressive peripheral neuropathy with abnormalities comparable to those associated with CMTX, thus providing proof of the critical role of Cx32 in the maintenance of peripheral nerve myelin and an animal model for CMTX. Frequently observed features include abnormally thin myelin sheaths, cellular onion bulb formation reflecting myelin degeneration-induced Schwann cell proliferation, and enlarged periaxonal collars while nerve conductance properties are altered only slightly. These observations are consistent with earlier hypotheses suggesting a function of Cx32 as a channel-forming protein that facilitates the communication between the abaxonal and adaxonal aspects of Schwann cell cytoplasm.

Fig. 2.

Schematic representation of the number of onion bulbs (A) and abnormally enlarged periaxonal collars (B, C) in femoral nerves of Cx32-deficient and wild-type mice at 4 weeks, 4 and 6 months, and 1 year of age. a, Note that onion bulbs develop only in the quadriceps nerve of Cx32-deficient mice (Cx32/Q) older than 4 months, whereas such structures are absent or found only very rarely in the saphenous nerve of the mutant (Cx32/S) and in the quadriceps (w.t./Q) and saphenous nerves (w.t./S) of the wild-type mice. *Significantly different compared with wild-type mice (p < 0.05); **significantly different compared with wild-type mice (p < 0.01).n indicates the number of mice investigated.B, C, At 6 months of age, both in quadriceps (B) and saphenous nerves (C), the number of abnormally enlarged periaxonal collars is elevated significantly in Cx32-deficient mice when compared with values from wild-type mice. The lack of significance in saphenous nerves of 1-year-old mice possibly can be explained by the large SDs. *Significantly different compared with wild-type mice (p < 0.05); **significantly different compared with wild-type mice (p < 0.01).n indicates the number of mice investigated.

Fig. 1.

Genotyping of Cx32 mutant mice. A, Partial restriction map of the Cx32 locus and schematic representation of the targeting construct and the targeting event. Homologous recombination resulted in an “in frame” insertion of the promoterless neo cassette and the concomitant disruption of the translated exon (exon 2). Exons are indicated as filled boxes. The neo cassette (NEO) is represented by an open box. E, EcoRI;P, PstI; S,SacII. Amplified PCR fragments are indicated with athick line. B, PCR analysis of the Cx32 locus and the targeting event in transgenic animals (control, no DNA added).

Fig. 3.

Electron migrographs of axon–Schwann cell units of wild-type (a) and connexin 32-deficient mice (b–g) at 4 (a, b) and 6 (e–g) months and 1 year of age (c, d). a, This axon (A) is enveloped by a sheath of compacted myelin (M). A basal lamina around the myelinating Schwann cell (arrow) and endoneurial collagen (C) separates the axon–Schwann cell unit from neighboring nerve fibers. b, This myelinated axon–Schwann cell unit is characterized by thin, compacted myelin surrounded by basal lamina-covered processes of Schwann cells forming an onion bulb (arrows). c, This onion bulb contains two myelinated axon–Schwann units.Large and small arrows point to a process and cell body of onion bulb cells, respectively. d, Axon–Schwann cell unit surrounded by processes of onion bulb cells (arrows). Note the expanded periaxonal collar containing vesicular inclusions (asterisks). e,f, Intimate interdigitations between the inner aspect of the Schwann cell and the axon (A). In e, the small arrows delineate a slender, loop-shaped protrusion of the axon (A) engulfing Schwann cell and/or axonal cytoplasm. In f, a cytoplasmic extension of the Schwann cell collar (arrow) protrudes into the axon.g, Abnormally organized noncompacted aspect of a myelinating Schwann cell (asterisk). Note the numerous vesicular inclusions and the expanded periaxonal collar of high electron density. Scale bars, 0.5 μm.