Peripheral myelin maintenance is a dynamic process requiring constant Krox20 expression

Abstract

Onset of myelination in Schwann cells is governed by several transcription factors, including Krox20/Egr2, and mutations affecting Krox20 result in various human hereditary peripheral neuropathies, including congenital hypomyelinating neuropathy (CHN) and Charcot-Marie-Tooth disease (CMT). Similar molecular information is not available on the process of myelin maintenance. We have generated conditional Krox20 mutations in the mouse that allowed us to develop models for CHN and CMT. In the latter case, specific inactivation of Krox20 in adult Schwann cells results in severe demyelination, involving rapid Schwann cell dedifferentiation and increased proliferation, followed by an attempt to remyelinate and a block at the promyelinating stage. These data establish that Krox20 is not only required for the onset of myelination but that it is also crucial for the maintenance of the myelinating state. Furthermore, myelin maintenance appears as a very dynamic process in which Krox20 may constitute a molecular switch between Schwann cell myelination and demyelination programs.

Figure 1.

Conditional excision of the floxed Krox20 allele. a, Schematic representation of the different Krox20 alleles, including the following: wild-type, Krox20^lacZ, Krox20^Cre, floxed (Krox20^flox), and deleted (Krox20^Δ) alleles. loxP sites are indicated by black arrowheads. Arrows indicate the positions of the primers used for PCR amplification, and the sizes of the amplified fragments are shown above the fragments. Primers p3 and p4 produce 160 and 195 bp fragments from wild-type and Krox20^flox alleles, respectively; primers p2 and p3 amplify a 210 bp fragment from the Krox20^Δ allele. E, EcoRI; S, SpeI. b–d, PCR analysis of sciatic nerve DNA from P1, P4, and P28 Krox20^Cre/flox mutants (b), extracted 28 d after injection from intraperitoneally injected TM (two rounds of 5 d injections) (c) or extracted 3, 8, and 28 d after injection from focally OHT- and DMSO-injected animals with the indicated genotypes (d). The excision of the second exon is demonstrated by the appearance of the 210 bp PCR fragment (deleted allele) and the relative reduction of the 195 bp fragment (Krox20^flox allele) compared with the 160 bp fragment (lacZ or Cre allele). Note the approximate twofold relative reduction of the floxed allele from P4 in Krox20^Cre/flox animals (b) and the detection of the deleted allele as early as 3 d after focal OHT injection (d). The letters above the lanes in d refer to the position of the nerve sample used for DNA extraction with respect to the site of injection: P, proximal, 5-mm-long pieces centered on the injection site; M, medial, 5 mm away from the injection site; D, distal, 10 mm away from the injection site. Control mice without Cre (Krox20^lacZ/flox, R26; data not shown), or injected with DMSO only, do not show second exon excision. e, f, Krox20 Western blotting analysis in protein extracts prepared from sciatic nerves from wild-type (WT) and Krox20^Cre/flox P1, P4, P10, and P28 animals (e) and Krox20^lacZ/flox and Krox20^lacZ/flox, R26-CreER^T animals (f), intraperitoneally TM or focally OHT injected and analyzed 3, 8, or 28 d after the last injection. e, Note the progressive decrease in Krox20 protein level in Krox20^cre/flox (Mut) compared with wild-type (WT) animals from P1 (no decrease), P4 (50% reduction), P10 (50% reduction), and P28 (90% reduction) as estimated by the relative intensities of the bands. f, Twenty-eight days after intraperitoneal TM injection, the analysis of three Krox20^lacZ/flox, R26-CreER^T animals revealed 40, 60, and 40% reductions in Krox20 protein levels, respectively, compared with the control (Krox20^lacZ/flox). After focal OHT injection, Krox20 protein levels are reduced by ∼40% from 3 d after injection.

Figure 2.

Sciatic nerves from P28 Krox20^Cre/flox mice are devoid of myelin. a, b, Toluidine blue-stained transverse sections of sciatic nerve from wild-type (a) and Krox20^Cre/flox (b) mice. c–f, Electron micrographs of sciatic nerve transverse sections from Krox20^Cre/flox (c, d, f) and wild-type (e) mice, illustrating the absence of myelin in the mutant. Note the intense proliferation of basal membranes (d, arrow) around the axons and the increased number of mitochondria within the axonal cytoplasm (compare e, f) in the mutants. Scale bars: a, b, 25 μm; c, 1.25 μm; d–f, 0.5 μm. WT, Wild type.

Figure 3.

Inactivation of Krox20 by intraperitoneal TM injection leads to discrete demyelination. The Krox20^lacZ/flox, R26-CreER^T animals were given injections for 5 d (a–g) or twice for 5 d (h), as indicated in Materials and Methods, and killed 28 d after the last injection. a, Toluidine blue-stained semithin section revealing normal density of axonal fibers. b–g, Micrographs of ultrathin sections illustrating the observation of denuded axons (b, arrows), of macrophages that have entered the Schwann cell cytoplasm (c, arrow) leading to myelin disruption around the axon (a), of intense proliferation of basal membranes (d, arrows), of onion bulbs (e, arrows), of mesaxons starting to wrap the axon (f, arrow and inset showing a higher magnification of the area), and of myelin sheaths abnormally thin (g, arrow) compared with a normal myelinated axon (g, arrowhead) [note the presence of completely denuded axons (a)]. These latter characteristics suggest that remyelination is occurring. h, Larger groups of denuded axons (encircled by the dotted line) are observed after double TM treatment. Scale bars: a, 25 μm; b, h, 2.5 μm; c–g, 0.5 μm.

Figure 4.

Inactivation of Krox20 by focal injection of OHT in the sciatic nerve leads to massive demyelination. a, b, Semithin, transverse toluidine blue-stained sections of carrier (a) and OHT-injected (b) sciatic nerves analyzed 8 d after injection. Severe myelin depletion is observed in the right half of the OHT-injected nerve. c–f, Electron microscopy analysis of OHT-injected sciatic nerve prepared 3 d (c), 8 d (d), or 28 d (e, f) after injection. c, Three days after the injection, myelin presents aspect of degradation (arrowhead) and some denuded axons are detected (arrows). d, Eight days after OHT injection, some areas are primarily devoid of myelin, with numerous denuded axons (arrows) and presence of macrophages (filled arrowhead) and of outfolded myelin (open arrowhead). Discrete cytoplasmic disorganizations in bundles of unmyelinated fibers (Remak) is occasionally observed in both experimental (c, d) and control animals and is likely to result from tissue fixation. e, f, Twenty-eight days after the injection, onion-bulb formations (e, arrowheads) are observed and numerous axons remain denuded (e, arrows), whereas others are wrapped with abnormally thin myelin sheaths (f, arrows). All of these observations were performed in the region proximal to the injection site (see Fig. 1b). Scale bars: a, b, 50 μm; e, 5 μm; c, d, f,1 μm.

Figure 5.

Krox20 inactivation results in transient and long-term expression of Sox2 and SCIP Schwann cell markers, respectively. Sciatic nerve sections were prepared 3, 8, or 28 d after DMSO or OHT injections of Krox20^lacZ/flox, R26-CreER^T mice and immunolabeled for Sox2 (green), a marker for immature Schwann cells, and SCIP (blue-green), a Schwann cell marker not expressed in mature myelinating cells. Cell nuclei were counterstained with Hoechst 33342 (dark blue). Note that OHT injection leads to rapid and transient accumulation of Sox2, whereas SCIP induction occurs later (8 d) but lasts longer. Scale bar, 55 μm.

Figure 6.

Krox20 inactivation results in a transient increase in Schwann cell proliferation. Cell proliferation was estimated by BrdU incorporation. The animals were given intraperitoneal injections of BrdU, the incorporation of which in DNA was subsequently revealed by immunostaining of sciatic nerve sections. Nuclei were counterstained with Hoechst 33342 or Sytox Green. a, b, BrdU incorporation 8 d after DMSO (a) and OHT (b) injections into the sciatic nerve of Krox20^lacZ/flox, R26-CreER^T mice. Nuclei are visualized with Sytox green (green), and those in S phase (arrows) are labeled with anti-BrdU antibodies (red). Scale bar, 75 μm. c, Estimation of the percentage of BrdU-positive nuclei in sections of the sciatic nerves from noninjected (control), DMSO-injected, and OHT-injected Krox20^lacZ/flox, R26-CreER^T mice 3, 8, and 28 d after injection. Quantitations were performed from lesioned areas (see Materials and Methods). The sections were derived from the proximal (P), medial (M), and distal (D) areas respective to the site of injection, as described in the legend to Figure 1b. Note that OHT treatment significantly increases the percentage of BrdU-positive cells within 3 d in all areas and that this level progressively returns to normal within 28 d. Values are means ± SD of at least three mice. Statistical significance was analyzed using the Student's t test: *p < 0.02; **p < 0.01.