Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves.
Myelinophagy was positively regulated by the Schwann cell JNK/c-Jun pathway, a central regulator of the Schwann cell reprogramming induced by nerve injury.We also present evidence that myelinophagy is defective in the injured central nervous system.These results reveal an important role for inductive autophagy during Wallerian degeneration, and point to potential mechanistic targets for accelerating myelin clearance and improving demyelinating disease.
Affiliation: Department of Cell and Developmental Biology, University College London, London WC1E 6BT, England, UK.
- Myelin Sheath/pathology*/physiology
- Peripheral Nerve Injuries/enzymology/pathology*
- Cells, Cultured
- JNK Mitogen-Activated Protein Kinases/metabolism
- Lipid Metabolism
- Mice, Transgenic
- Proto-Oncogene Proteins c-jun/metabolism
- Sciatic Nerve/pathology
- TOR Serine-Threonine Kinases/metabolism
- Wallerian Degeneration/pathology
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fig6: Myelinophagy is mTOR independent and promoted by lithium and ceramide. (A) Western blot showing increased expression of pmTOR, p-S6, and p-AKT in cut WT nerves. Quantification of Western blots is shown in Fig. S5 A. (B) Immunolabeling showing regulation of MPZ breakdown by rapamycin, ceramide, or JNK inhibitor SP600125 in Schwann cell cultures (from P8 mice animals, treated 3 d in vitro). Myelin breakdown is unchanged by rapamycin, reduced by ceramide, and increased by JNK inhibitor compared with untreated cultures. (C) Graph showing MPZ+ myelin area in Schwann cell cultures (as in Fig. 6 B) after different treatments. Data are presented as mean ± SEM (error bars) from three independent experiments with a minimum of 200 cells analyzed per condition/experiment. *, P < 0.05; **, P < 0.01 (treated cells relative to untreated controls). (D) Electron micrographs showing fewer intact myelin sheaths in nerve segments maintained in vitro for 4 d in the presence of ceramide and lithium, compared with control cultures. The graph shows quantification of the number of intact myelin sheaths in control segments and segments treated with ceramide and lithium. Data are presented as mean ± SEM (error bars) from three independent experiments with a minimum of 10 picture frames analyzed per condition/experiment. *, P < 0.05; **, P < 0.01 (treated cells relative to untreated controls). (E) Western blot showing increased LC3 II accumulation in nerve segments maintained in vitro for 3 d and treated with ceramide or lithium in the presence or absence of NH4Cl (3 h treatment). The graph shows increased net LC3 II flux after ceramide and lithium treatment compared with control cultures. Data are presented as mean ± SEM (error bars) from three independent experiments. *, P < 0.05 (treated cells relative to untreated controls). (F) Graph showing quantification of MPZ+ area in control Schwann cell cultures (WT) and cultures in which autophagy was blocked (Atg7 cKO cultures). The increased myelin degradation of MPZ seen after treatment with ceramide and lithium in WT dissociated Schwann cell cultures is blocked in Atg7 cKO cultures. See Fig. S5 D for pictures of immunolabeling. Data are presented as mean ± SEM (error bars) from three independent experiments with a minimum of 10 picture frames analyzed per condition/experiment. *, P < 0.05; ns, not significant (treated cells relative to untreated controls).
In starvation autophagy, the best-studied model of autophagy regulation, autophagy is activated by reduction in the activity of the autophagy inhibitor mTOR (Ravikumar et al., 2010). Therefore, we examined levels of pmTOR, and pAKT and pS6, which are upstream and downstream markers for mTOR, respectively. While uninjured nerves showed low levels of pmTOR, pS6, and pAKT, these were significantly up-regulated at 5 and 7 d after transection (Fig. 6 A). In addition, neither mTOR activation by starvation (serum deprivation) nor mTOR inhibition by rapamycin altered the rate of myelin breakdown in Schwann cell cultures (Fig. 6, B and C). This was confirmed by analysis of myelin sheath collapse and measurements of autophagy flux in cultured nerve segments treated with rapamycin (Fig. S5, B and C). Treatment with insulin or neuregulin, stimulators of mTOR, also failed to affect myelin protein breakdown (Fig. 6 C). Together this shows that down-regulation of mTOR does not provide the signal for activation of Schwann cell autophagy.