N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination

Abstract

Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.

Fig. 1.

N-WASp expression is developmentally regulated in Schwann cells and its conditional deletion is associated with motor impairment. (A) Confocal images of sciatic nerve transverse sections from wild-type mouse reveal N-WASp to be localized around individual axons in a pattern distinct from those of S100β and DAPI. (B) N-WASp immunofluorescence increases between P1 and P5, diminishes by P13 and remains low at P28. (C) qRT-PCR analysis shows changes over time in N-WASp (left) and Mbp (right) mRNA levels in wild-type sciatic nerves (mean of three independent experiments expressed relative to Gapdh + s.d.). (D) Two-month-old (left) and 7-month-old (right) mutant mice have a wider and outward-pointing hindpaw stance. Footprint patterns recorded from forepaws (red) and hindpaws (blue) reveal a widened ataxic gait compared with the narrow, even stride of controls (see Movies 1 and 2 in the supplementary material). (E) Rotarod tests performed on three consecutive days (three trials per day) showing significantly reduced mean (± s.e.m.) hold times in 2-month-old mutant mice. (F) Tremor activity in 2-month-old mutant mice shown as a representative recording of movement amplitude and a graph indicating numbers of tremor events at each frequency (mean ± s.e.m.) (data representative of three independent experiments).

Fig. 2.

N-WASp is required for Schwann cell myelination. (A-C) Light microscopy of semi-thin transverse Toluidine Blue-stained sections from control and mutant P5 (A) and P60 (B) sciatic nerves and P60 L3 ventral roots (C). At P5, axons in control nerves are sorted and many have myelin sheaths, whereas in mutant samples bundles of unsorted axons remain within Schwann cell families. Asterisks indicate unsorted axon bundles. At P60, all large caliber axons in control nerves are myelinated, whereas in the mutant only rare myelinated fibers are observed amongst ensheathed amyelinated axons (see Table S1 in the supplementary material). (D-G) Electron micrographs of control (D) and mutant (E-G) fibers in P60 samples. Although axon membranes are covered completely in both control and mutant samples, mutant Schwann cells do not show spiral membrane wrapping. Where mesaxons abut, they show little evidence of further circumferential extension (F), often elaborating multiple short interdigitating processes (G). In both mutant and control nerves, all fibers are surrounded by basal lamina and the interfiber space is richly invested with collagen fibrils. (H) Immunofluorescence analysis of control and mutant Schwann cells co-cultured with control dorsal root ganglia neurons co-stained with DAPI and antibodies to neurofilament and Mbp. The numbers of DAPI-stained nuclei and neurofilament-stained axons are equivalent in both culture types, but mutant cells do not produce Mbp-stained internodes (data representative of five independent experiments).

Fig. 3.

Increased number and proliferation of N-WASp-deficient Schwann cells. (A) Haematoxylin- and Eosin-stained sciatic nerve transverse sections show increased numbers of Schwann cell nuclei in mutant nerves at all timepoints studied (bar chart). (B,C) The number of proliferating cells is increased in mutant compared with control nerve sections as assessed by DAPI and Ki67 staining at P5 and P28 (B) and by BrdU staining of cells 24 hours after BrdU intraperitoneal injection at P5 and P24 (C). Data are expressed as mean + s.e.m. of at least four independent experiments.

Fig. 4.

Myelin gene expression and lamellipodia formation are impaired in N-WASp-deficient Schwann cells. (A) Western blot analysis reveals diminished myelin protein accumulation in mutant P10 sciatic nerves. (B) qRT-PCR analysis also shows reduced expression of myelin-related genes in the N-WASp^−/− sciatic nerves (values are expressed relative to Gapdh transcript levels and represent mean + s.d. of three independent experiments). (C) Confocal images showing absence of Mbp staining in nerves from P5 and P28 mutants. (D) Western blot showing modest increases in expression of Oct6, Krox20 and Sox10 in mutant relative to control P10 sciatic nerves. (E) Phase-contrast micrograph showing reduced lengths of the filamentous processes extended by mutant compared with control Schwann cells cultured over laminin 2. (F) Immunofluorescence analysis showing lack of well-formed F-actin-enriched process tips in mutant compared with control FITC-phalloidin-stained Schwann cells. (G) Immunofluorescence analysis showing that the numbers of axial lamellipodia (white arrows) present at the terminus of the Schwann cell long axis are equivalent in control and mutant Schwann cells, whereas the number of radial lamellipodia (red arrows) formed outside this region is dramatically reduced in the mutant samples. (E-G) Measurements of process length, number of process tips with intact leading edges and number of processes per cell are mean + s.d. of 100 representative Schwann cells per sample.