Impaired differentiation of Schwann cells in transgenic mice with increased PMP22 gene dosage

Abstract

An intrachromosomal duplication containing the PMP22 gene is associated with the human hereditary peripheral neuropathy Charcot-Marie-Tooth disease type 1A, and PMP22 overexpression as a consequence of increased PMP22 gene dosage has been suggested as causative event in this frequent disorder of peripheral nerves. We have generated transgenic mice that carry additional copies of the pmp22 gene to prove that increased PMP22 gene dosage is sufficient to cause PNS myelin deficiencies. Mice carrying approximately 16 and 30 copies of the pmp22 gene display a severe congenital hypomyelinating neuropathy as characterized by an almost complete lack of myelin and marked slowing of nerve conductions. Affected nerves contain an increased number of nonmyelinating Schwann cells, which do not form onion bulbs but align in association with axons. The mutant Schwann cells are characterized by a premyelination-like state as indicated by the expression of embryonic Schwann cell markers. Furthermore, continued Schwann cell proliferation is observed into adulthood. We hypothesize that Schwann cells are impaired in their differentiation into the myelinating phenotype, leading to a disorder comparable to severe cases of hereditary motor and sensory neuropathies. Our findings, combined with the analysis of heterozygous and homozygous PMP22-deficient mice, indicate that aberrant pmp22 gene copy numbers cause various forms of myelination defects.

Fig. 1.

Generation of PMP22-transgenic mice.A, Structure of the genomic PMP22 cosmid clone pTCF-6.1. Identified exons are indicated by filled boxes and numbered below; cosmid-derived sequences are represented by open boxes. The DNA probes used for identification and characterization of the clone (3′- and 5′-probes) are indicated bythick lines. The 43-kb-long NruI fragment of pTCF-6.1 was purified on a 0.6% agarose gel, isolated by electroelution, and microinjected into fertilized oocytes.S, SalI; K,KpnI; B, BamHI;N, NruI. B, Southern blot analysis of BamHI-digested genomic DNA of PMP22-transgenic and wild-type founder mice using the 5′-probe (lanes 2 and 3 are wild type, andlanes 1, 4, and 5 represent the transgenic lines TgN247, TgN248, and TgN249, respectively).

Fig. 2.

Analysis of the expression of PMP22 and P0. Northern blot analysis of PMP22-transgenic and wild-type mice (A–C). The same blot was hybridized first with a PMP22 cDNA probe (A) and subsequently with P0 cDNA probe (B). Ethidium bromide-stained agarose gel (C; 18S RNA) is shown as quantitation control. RNA was isolated from heart (lanes 1–4) and from sciatic nerves (lanes 5 and 6) of 21-d-old PMP22-transgenic mice (lanes 1, 3, and 5) and wild-type (lanes 2, 4, and 6) siblings (note that the exposure times of the different blots and probes were not identical). Western blot analysis of PMP22-transgenic and wild-type mice (D). Crude sciatic nerve homogenates (20 μg of protein) of PMP22-transgenic mice (lanes 1and 3; TgN248) and wild-type littermates (lanes 2 and 4) were separated on 12.5% SDS-PAGE and blotted to nitrocellulose membranes. Proteins were probed with antibodies specific for PMP22 (lanes 1 and2) or P0 (lanes 3 and4).

Fig. 3.

Nerve conduction analysis of PMP22-transgenic mice and wild-type littermates. Top, Summary of the results of the nerve conduction studies in the facial (left) and sciatic nerves (right; Mean ± SD;n = number of animals analyzed) of 10-week-old PMP22-transgenic and wild-type control mice. We were unable to elicit proximal sciatic nerve CMAP responses in two PMP22-transgenic mice. Therefore, the conduction velocity in the sciatic nerve could be determined in only three mice of this group and may therefore be slightly overestimated. Bottom, Redrawn original recordings of CMAP responses after stimulation of the facial nerve (left) and the distal sciatic nerve (right) of a 79-d-old control mouse (top row) and of a 79-d-old PMP22-transgenic sibling (bottom row). Note the different time and amplitude scales. Recordings were reproducible on repeated stimulation.

Fig. 4.

Assessment of myelination in semithin sections and immunohistological analysis. Cross-sections (2 μm) of 21-d-old wild-type (a) and PMP22-transgenic mice (a′) were stained with toluidine blue. Note the lack of detectable myelin (a′). Immunohistological localization of the cell surface molecules LNGFR (b,b′), N-CAM (c, c′), and L1 (d, d′) in femoral quadriceps nerves of adult wild-type (b–d) and mutant mice (b′–d′). Although LNGFR is prominently upregulated in mutant mice (b, b′), N-CAM is moderately (c, c′) and L1 weakly increased (d, d′). Note that the nonmyelinating axon-Schwann cell units also are labeled stronger for N-CAM and L1 in the mutants (c′, d′) than in the wild-type mice (c, d). Arrow inc marks the weakly N-CAM-immunoreactive perineurium of a wild-type nerve. Scale bars, 50 μm.

Fig. 5.

Ultrastructural examination of sciatic nerves. Electron microscopy of sciatic nerves of 10- (a, b), 21- (c), and 72-d-old (d) wild-type (a) or PMP22-transgenic mice (b–d; TgN248). In 10-d-old wild-type mice (a), larger-caliber axons (A) are associated with compact myelin (M), whereas in transgenic littermates (b), larger-caliber axons are ensheathed by Schwann cells, which do not form myelin. Arrowhead points to Schwann cell basal lamina. In 21-d-old PMP22-transgenic mice (c), most of the larger-caliber axons are associated with Schwann cells, which do not form myelin; however, ∼10% of the axons having acquired a 1:1 ratio with Schwann cells are surrounded by a thin sheath of compact myelin (M). Note the prominent pockets of Schwann cell basal laminae (arrowheads), which are occasionally filled with Schwann cell processes of high electron density (double arrowheads). A′, Axons devoid of an ensheathing Schwann cell are found that are surrounded by pockets of basal laminae, indicative of the previous presence of a Schwann cell (arrowheads). In 72-d-old PMP22-transgenic mice (d), larger-caliber axons (A) are always associated with Schwann cells devoid of myelin. Profiles of supernumerary Schwann cell basal laminae (arrowheads) are seen consistently but appear less prominent than in 21-d-old mice. Scale bars, 1 μm.

Fig. 6.

Histological analysis of the quadriceps muscle of PMP22-transgenic and wild-type mice. Normal chessboard-like distribution pattern of types I (slow-twitch muscle,dark) and II (fast twitch muscle, light) muscle fibers of wild-type animals (A). PMP22-transgenic animals (B, C) show prominent segregation and grouping of types I and II fibers (B) and few hypertrophic fibers (arrows) but no significant atrophy. C, Late stage of neurogenic myopathy with type grouping and large groups of atrophic fibers. Scale bar, 25 μm.

Fig. 7.

Cell number in cross-sections of sciatic nerves. Sciatic nerves of 10-week-old wild-type mice (A) and PMP22-transgenic siblings (B) were analyzed histologically after DAPI staining. Scale bar, 50 μm. Ten micrometer cryosections of PMP22-transgenic and wild-type mice of various ages were stained with DAPI, and the nuclei were counted (C). The bars indicate the relative surplus of nuclei in the sciatic nerves of PMP22-transgenic mice compared with wild-type animals. p values (two-tailed Student’st test) are given.

Fig. 8.

Schwann cell proliferation in the sciatic nerve of PMP22-transgenic and wild-type mice. Longitudinal sections (2 μm) of BrdU-labeled sciatic nerves of 21-d-old wild-type mice (A) and PMP22-transgenic littermates (B). Frequent incorporation of BrdU in PMP22-transgenic mice indicates proliferation of Schwann cells (arrows inB), whereas only rare signals in the wild type are detectable. Scale bar, 25 μm. Ultrathin longitudinal section of a sciatic nerve of a 10-week-old PMP22-transgenic mouse (C). A large caliber axon (black asterisks) is ensheathed by darkly appearing Schwann cell processes (arrows in C). White asterisks indicate Schwann cell perikarya with direct contact to the axon. Note that their frequency is unusually high. The two neighboring Schwann cell nuclei at the left possibly indicate that a Schwann cell mitosis takes place (arrowheads, endoneurial collagen). Scale bar, 5 μm.