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Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves.

Gomez-Sanchez JA, Carty L, Iruarrizaga-Lejarreta M, Palomo-Irigoyen M, Varela-Rey M, Griffith M, Hantke J, Macias-Camara N, Azkargorta M, Aurrekoetxea I, De Juan VG, Jefferies HB, Aspichueta P, Elortza F, Aransay AM, Martínez-Chantar ML, Baas F, Mato JM, Mirsky R, Woodhoo A, Jessen KR - J. Cell Biol. (2015)

Bottom Line: 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.

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Affiliation: Department of Cell and Developmental Biology, University College London, London WC1E 6BT, England, UK.

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Pharmacological block of autophagy prevents myelin degradation. (A) Western blot showing a block in degradation of the myelin proteins MPZ and MBP in nerve segments maintained in vitro for 5 d and treated with different pharmacological inhibitors: untreated, lysosomal inhibitor, NH4Cl, and autophagy inhibitors 3-MA and bafilomycin A1 (Baf). No such effect is seen in freshly isolated nerve segments treated with these inhibitors for 3 h. The black line indicates that intervening lanes have been spliced out. Graphs show densitometric quantification of Western blots. Data are presented as mean ± SEM (error bars) from three independent experiments. n.s., nonsignificant; *, P < 0.05. (B) qPCR analysis showing no significant differences (n.s.) in mRNA levels of Mpz and Mbp in nerve segments maintained in vitro for 5 d and treated with 3-MA and Baf, compared with untreated segments. Data are expressed as log2 fold change in 5D cultured nerve segments relative to uncut nerves. n = 3 for each condition. Data are presented as mean ± SEM (error bars). (C) Electron micrographs showing abundant intact myelin sheaths in nerve segments cultured in vitro for 4 d in the presence of 3-MA, compared with untreated cultures. The graph shows a quantification of the number of intact myelin sheaths in untreated segments and segments treated with 3-MA. (D) Teased fibers from untreated and 3-MA treated nerve segments (5 d) stained with FluoroMyelin red. The graph shows a quantification of the myelin fluorescent area. (E) Immunolabeling showing a block in the degradation of lipids (Bodipy+ cells) in dissociated Schwann cell cultures after 3 d of 3-MA treatment. Graph shows quantification of Bodipy+ area. (F) 3-MA blocks myelin protein degradation in dissociated Schwann cell cultures. Immunolabeling showing a block in degradation of MPZ in dissociated Schwann cells (S100+) cultured for 5 d in the presence of 3-MA. The graph shows MPZ+ area in dissociated cultures treated with 3-MA. (G) Graph showing the number of MPZ+ Schwann cells in dissociated cultures treated for 5 d with 3-MA, bafilomycin, and NH4Cl. (C–G) 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).
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fig3: Pharmacological block of autophagy prevents myelin degradation. (A) Western blot showing a block in degradation of the myelin proteins MPZ and MBP in nerve segments maintained in vitro for 5 d and treated with different pharmacological inhibitors: untreated, lysosomal inhibitor, NH4Cl, and autophagy inhibitors 3-MA and bafilomycin A1 (Baf). No such effect is seen in freshly isolated nerve segments treated with these inhibitors for 3 h. The black line indicates that intervening lanes have been spliced out. Graphs show densitometric quantification of Western blots. Data are presented as mean ± SEM (error bars) from three independent experiments. n.s., nonsignificant; *, P < 0.05. (B) qPCR analysis showing no significant differences (n.s.) in mRNA levels of Mpz and Mbp in nerve segments maintained in vitro for 5 d and treated with 3-MA and Baf, compared with untreated segments. Data are expressed as log2 fold change in 5D cultured nerve segments relative to uncut nerves. n = 3 for each condition. Data are presented as mean ± SEM (error bars). (C) Electron micrographs showing abundant intact myelin sheaths in nerve segments cultured in vitro for 4 d in the presence of 3-MA, compared with untreated cultures. The graph shows a quantification of the number of intact myelin sheaths in untreated segments and segments treated with 3-MA. (D) Teased fibers from untreated and 3-MA treated nerve segments (5 d) stained with FluoroMyelin red. The graph shows a quantification of the myelin fluorescent area. (E) Immunolabeling showing a block in the degradation of lipids (Bodipy+ cells) in dissociated Schwann cell cultures after 3 d of 3-MA treatment. Graph shows quantification of Bodipy+ area. (F) 3-MA blocks myelin protein degradation in dissociated Schwann cell cultures. Immunolabeling showing a block in degradation of MPZ in dissociated Schwann cells (S100+) cultured for 5 d in the presence of 3-MA. The graph shows MPZ+ area in dissociated cultures treated with 3-MA. (G) Graph showing the number of MPZ+ Schwann cells in dissociated cultures treated for 5 d with 3-MA, bafilomycin, and NH4Cl. (C–G) 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).

Mentions: If Schwann cells degrade myelin by autophagy, autophagy inhibitors should reduce myelin breakdown. We therefore cultured nerve segments with the widely used autophagy inhibitors 3-methyladenine (3-MA) and Bafilomycin A1 (Klionsky et al., 2012). The lysosomal inhibitor NH4Cl was included for comparison. These inhibitors strikingly prevented the degradation of the myelin proteins MPZ and MBP in nerve segments maintained in vitro for 5 d but not in uncut nerve segments, indicating that autophagy is required for myelin protein breakdown (Fig. 3 A). This was not due to transcriptional changes, as we did not find significant differences in mRNA levels in nerve segments treated with these autophagy inhibitors (Fig. 3 B). Histologically, we found that 3-MA reduced myelin collapse in cultured nerves compared with control nerves, in which most sheaths appeared as collapsed whorls (Fig. 3 C).


Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves.

Gomez-Sanchez JA, Carty L, Iruarrizaga-Lejarreta M, Palomo-Irigoyen M, Varela-Rey M, Griffith M, Hantke J, Macias-Camara N, Azkargorta M, Aurrekoetxea I, De Juan VG, Jefferies HB, Aspichueta P, Elortza F, Aransay AM, Martínez-Chantar ML, Baas F, Mato JM, Mirsky R, Woodhoo A, Jessen KR - J. Cell Biol. (2015)

Pharmacological block of autophagy prevents myelin degradation. (A) Western blot showing a block in degradation of the myelin proteins MPZ and MBP in nerve segments maintained in vitro for 5 d and treated with different pharmacological inhibitors: untreated, lysosomal inhibitor, NH4Cl, and autophagy inhibitors 3-MA and bafilomycin A1 (Baf). No such effect is seen in freshly isolated nerve segments treated with these inhibitors for 3 h. The black line indicates that intervening lanes have been spliced out. Graphs show densitometric quantification of Western blots. Data are presented as mean ± SEM (error bars) from three independent experiments. n.s., nonsignificant; *, P < 0.05. (B) qPCR analysis showing no significant differences (n.s.) in mRNA levels of Mpz and Mbp in nerve segments maintained in vitro for 5 d and treated with 3-MA and Baf, compared with untreated segments. Data are expressed as log2 fold change in 5D cultured nerve segments relative to uncut nerves. n = 3 for each condition. Data are presented as mean ± SEM (error bars). (C) Electron micrographs showing abundant intact myelin sheaths in nerve segments cultured in vitro for 4 d in the presence of 3-MA, compared with untreated cultures. The graph shows a quantification of the number of intact myelin sheaths in untreated segments and segments treated with 3-MA. (D) Teased fibers from untreated and 3-MA treated nerve segments (5 d) stained with FluoroMyelin red. The graph shows a quantification of the myelin fluorescent area. (E) Immunolabeling showing a block in the degradation of lipids (Bodipy+ cells) in dissociated Schwann cell cultures after 3 d of 3-MA treatment. Graph shows quantification of Bodipy+ area. (F) 3-MA blocks myelin protein degradation in dissociated Schwann cell cultures. Immunolabeling showing a block in degradation of MPZ in dissociated Schwann cells (S100+) cultured for 5 d in the presence of 3-MA. The graph shows MPZ+ area in dissociated cultures treated with 3-MA. (G) Graph showing the number of MPZ+ Schwann cells in dissociated cultures treated for 5 d with 3-MA, bafilomycin, and NH4Cl. (C–G) 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).
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fig3: Pharmacological block of autophagy prevents myelin degradation. (A) Western blot showing a block in degradation of the myelin proteins MPZ and MBP in nerve segments maintained in vitro for 5 d and treated with different pharmacological inhibitors: untreated, lysosomal inhibitor, NH4Cl, and autophagy inhibitors 3-MA and bafilomycin A1 (Baf). No such effect is seen in freshly isolated nerve segments treated with these inhibitors for 3 h. The black line indicates that intervening lanes have been spliced out. Graphs show densitometric quantification of Western blots. Data are presented as mean ± SEM (error bars) from three independent experiments. n.s., nonsignificant; *, P < 0.05. (B) qPCR analysis showing no significant differences (n.s.) in mRNA levels of Mpz and Mbp in nerve segments maintained in vitro for 5 d and treated with 3-MA and Baf, compared with untreated segments. Data are expressed as log2 fold change in 5D cultured nerve segments relative to uncut nerves. n = 3 for each condition. Data are presented as mean ± SEM (error bars). (C) Electron micrographs showing abundant intact myelin sheaths in nerve segments cultured in vitro for 4 d in the presence of 3-MA, compared with untreated cultures. The graph shows a quantification of the number of intact myelin sheaths in untreated segments and segments treated with 3-MA. (D) Teased fibers from untreated and 3-MA treated nerve segments (5 d) stained with FluoroMyelin red. The graph shows a quantification of the myelin fluorescent area. (E) Immunolabeling showing a block in the degradation of lipids (Bodipy+ cells) in dissociated Schwann cell cultures after 3 d of 3-MA treatment. Graph shows quantification of Bodipy+ area. (F) 3-MA blocks myelin protein degradation in dissociated Schwann cell cultures. Immunolabeling showing a block in degradation of MPZ in dissociated Schwann cells (S100+) cultured for 5 d in the presence of 3-MA. The graph shows MPZ+ area in dissociated cultures treated with 3-MA. (G) Graph showing the number of MPZ+ Schwann cells in dissociated cultures treated for 5 d with 3-MA, bafilomycin, and NH4Cl. (C–G) 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).
Mentions: If Schwann cells degrade myelin by autophagy, autophagy inhibitors should reduce myelin breakdown. We therefore cultured nerve segments with the widely used autophagy inhibitors 3-methyladenine (3-MA) and Bafilomycin A1 (Klionsky et al., 2012). The lysosomal inhibitor NH4Cl was included for comparison. These inhibitors strikingly prevented the degradation of the myelin proteins MPZ and MBP in nerve segments maintained in vitro for 5 d but not in uncut nerve segments, indicating that autophagy is required for myelin protein breakdown (Fig. 3 A). This was not due to transcriptional changes, as we did not find significant differences in mRNA levels in nerve segments treated with these autophagy inhibitors (Fig. 3 B). Histologically, we found that 3-MA reduced myelin collapse in cultured nerves compared with control nerves, in which most sheaths appeared as collapsed whorls (Fig. 3 C).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell and Developmental Biology, University College London, London WC1E 6BT, England, UK.

Show MeSH
Related in: MedlinePlus