Arrest of myelination and reduced axon growth when Schwann cells lack mTOR

Abstract

In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell.

Figure 1.

Conditional inactivation of mTOR in Schwann cells. A, PCR of genomic DNA isolated from mTOR^flox/flox and mutant quadriceps nerves shows efficient recombination in mutant nerves. B, Immunofluorescence analysis of P21 teased individual quadriceps nerve fibers confirms the loss of mTOR from the cytoplasm of Schwann cells in the mutant. The Schwann cell nucleus stained with TOTO-3 is in blue and mTOR is in green. Scale bar, 10 μm. C, Western blot analysis of lysates from P21 sciatic nerve shows that mTOR protein is reduced in the mutant and that the mTOR downstream targets S6 and 4E-BP1 are less phosphorylated. In contrast, there is a substantial increase in phosphorylation at T308 of Akt, and no decrease in phosphorylation at S473 of Akt. Gamma-actin was the loading control. D, Immunofluorescence staining of P21 teased quadriceps nerve fibers confirmed the loss of phospho (P)-S6 in Schwann cells lacking mTOR. Myelin was visualized using antibodies against Po in red, phospho-S6 in green, and Schwann cells nuclei were stained with TOTO-3. Scale bar, 10 μm. E, Nerve conduction velocities in the quadriceps nerves of control and mutant mice are represented in a vertical scatter plot. Each point is the mean of measurements from both nerves from one mouse; eight mice were analyzed. Horizontal lines are the SEMs and p < 0.005. F, Rotarod testing was at two speeds of rotation; mutant mice were significantly worse at both speeds. Values are expressed as mean time to fall ± SEM (n = 6, *p < 0.05, **p < 0.005).

Figure 2.

Mutant Schwann cells sort axons and differentiate, but their nerves are abnormally small. A, Electron micrographs of cross-sections of quadriceps nerves from floxed mTOR and mutant mice at P2, a time when axon sorting is normally well advanced, showed that formation of 1:1 promyelinating figures was normal in mutant mice. Scale bar, 5 μm. B, C, Immunofluorescence staining of longitudinal sections of quadriceps nerves with antibodies against Sox10 (for all Schwann cells) and Krox20 (transcription factor required for progression beyond the promyelinating stage) and quantitation revealed no difference in double-labeled Schwann cells between floxed mTOR and mutant mice at P21, showing that loss of mTOR did not prevent Schwann cell differentiation. Scale bar, 20 μm. D, Western blot of sciatic nerve lysates showing no decrease in Krox20, Dlg1, or the active cleaved fragment of axonal Nrg1 in the mutant but reduced amounts of the myelin protein P0. Gamma-actin was the loading control. E, F, Electron micrographs of quadriceps nerves at P21 show that the cross-sectional area of mutant nerves is much smaller than normal, which may explain why the density of Schwann cells revealed by Sox10 staining in the mutant shown in C appears to be increased. The decreased cross-sectional area was first significant at P7 and became more pronounced with age (means ± SEM, n ≥ 3, **p < 0.01, ***p < 0.001). Scale bar, 20 μm.

Figure 3.

Myelination is delayed in the absence of mTOR in Schwann cells. A, B, Electron microscopy revealed a large number of sorted but unmyelinated, promyelinating profiles at P21. When quantitated, the difference in the number of promyelinated profiles as a percentage of all sorted axons was significantly higher in mutant quadriceps nerves from P2 to P42 (means ± SEM, n ≥ 3, **p < 0.01, ***p < 0.001). However, by P90, the percentage of unmyelinated axons in the mutant had declined to 5%. Scale bar, 2 μm. C, Vertical scatter plots of quadriceps axonal diameters for promyelination-stage axons in three independent control mice (1–3) and three mutant mice (4–6) at P42. All the control values were <1.3 μm. However, in the mutant, many large axons were sorted into a 1:1 relationship with Schwann cells that had still not myelinated (4–6). The median values for animals 4, 5, and 6 were 1.7, 1.9, and 1.9 μm respectively.

Figure 4.

Myelin produced by mTOR mutant Schwann cells is thinner than normal and stays thin during development. Electron micrographs of cross-sections of quadriceps nerves shows sorted axons in mutant nerves but thin myelin from P7 to P90. Scale bar, 5 μm.

Figure 5.

Mutant nerves have thin myelin and reduced axon diameters. A, B, Quantitation of g-ratios as a function of axon diameter from P14 to P90 indicated strong diversion of the regression lines through the data for control compared with mutant quadriceps nerves, which was confirmed by analysis of mean values ± SEM (n ≥ 3). At each age, there was a significant difference between control and mutant (**p < 0.05, ***p < 0.005). However, there was no significant difference between the g-ratios measured at P14 and P90 for control nerves or between g-ratios measured at P14 and P90 for mutant nerves. C, Panel A indicated that there were fewer large caliber axons in the mutant at older ages. This was confirmed by measuring mean axonal diameters ± SEM (n ≥ 3) for control and mutant. A significant reduction in axonal diameter in the mutant was found from P21 onwards (**p < 0.01).

Figure 6.

Mutant nerves have reduced neurofilament phosphorylation and high neurofilament packing density. A, At P21, there is a significant difference in axonal caliber between control and mutant quadriceps axons (Fig. 5C). Western blotting of sciatic nerve lysates at the same age showed a major decrease in the extent of neurofilament H phosphorylation, as determined by the antibody SMI-32, which recognizes an epitope normally masked by phosphorylation and which is uncovered on dephosphorylation. Other phosphorylated epitopes recognized by the antibody SMI-34 were unaffected, and MAG was not reduced. Tubulin and actin were loading controls. B, Electron micrograph of mutant quadriceps at P90 shows the increased density within a promyelinating axon (diameter, 3.1 μm beside a myelinated axon of comparable diameter, 3.3 μm). Scale bar, 1 μm.

Figure 7.

Mutant nerves have shorter internodes and aberrant myelin. A, B, Immunofluorescence imaging (A) of a teased mutant fiber using an antibody against radixin (green), which is concentrated at microvilli, and TOTO-3 to visualize Schwann cell nuclei (blue). Arrows indicate the position of nodes of Ranvier. This reveals some variability in internodal length, which is quantitated in the histogram (B) showing the frequency distribution of internodal length in control and mutant quadriceps nerve. Scale bar, 50 μm. C, D, Electron micrographs of aberrant profiles from mutant nerves observed at all ages from P90. Outfoldings of thin myelin are shown in C. Arrows point to redundant basal lamina with vacuolation in the Schwann cell cytoplasm surrounding an unmyelinated axon. Scale bars: C, 2 μm; D, 1 μm.