Anat Cell Biol.  2011 Mar;44(1):41-49. 10.5115/acb.2011.44.1.41.

Transient lysosomal activation is essential for p75 nerve growth factor receptor expression in myelinated Schwann cells during Wallerian degeneration

• Jung J
• Cai W
• Jang SY
• Shin YK
• Suh DJ
• Kim JK
• Park HT

• KMID: 2168867
• DOI: http://doi.org/10.5115/acb.2011.44.1.41

Abstract

Myelinated Schwann cells in the peripheral nervous system express the p75 nerve growth factor receptor (p75NGFR) as a consequence of Schwann cell dedifferentiation during Wallerian degeneration. p75NGFR has been implicated in the remyelination of regenerating nerves. Although many studies have shown various mechanisms underlying Schwann cell dedifferentiation, the molecular mechanism contributing to the re-expression of p75NGFR in differentiated Schwann cells is largely unknown. In the present study, we found that lysosomes were transiently activated in Schwann cells after nerve injury and that the inhibition of lysosomal activation by chloroquine or lysosomal acidification inhibitors prevented p75NGFR expression at the mRNA transcriptional level in an ex vivo Wallerian degeneration model. Lysosomal acidification inhibitors suppressed demyelination, but not axonal degeneration, thereby suggesting that demyelination mediated by lysosomes may be an important signal for inducing p75NGFR expression. Tumor necrosis factor-alpha (TNF-alpha) has been suggested to be involved in regulating p75NGFR expression in Schwann cells. In this study, we found that removing TNF-alpha in vivo did not significantly suppress the induction of both lysosomes and p75NGFR. Thus, these findings suggest that lysosomal activation is tightly correlated with the induction of p75NGFR in demyelinating Schwann cells during Wallerian degeneration.

Keyword

Schwann cells; Lysosomes; p75NGFR; Demyelination; Wallerian degeneration

MeSH Terms

Axons
Cell Dedifferentiation
Chloroquine
Demyelinating Diseases
Lysosomes
Myelin Sheath
Nerve Growth Factor
Peripheral Nervous System
RNA, Messenger
Schwann Cells
Tumor Necrosis Factor-alpha
Wallerian Degeneration
Chloroquine
Nerve Growth Factor
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 Transient induction of lysosomal associated membrane protein 1 (LAMP1) in demyelinating Schwann cells. (A) LAMP1 was localized in perinuclear areas (arrows) in control sciatic nerves. (B) Sciatic nerve crush-induced LAMP1 in demyelinating Schwann cells, particularly around myelin ovoids 3 days (3d) after injury. The upper panels are double staining against LAMP1 and S100 (arrow), and the lower panels are double staining against LAMP1 and myelin basic protein (MBP). Asterisk in the middle panels are myelin ovoids demonstrated by differential interference microscopy. Asterisks in the lower panels are MBP-positive clumps surrounded by LAMP1 immunoreactivity. Arrows indicate a LAMP1-negative intact myelin sheath. (C) Downregulation of LAMP1 in demyelinating Schwann cells 7 days (7d) after injury (asterisks). Arrows indicate LAMP1 and CD68-positive macrophages. Macrophages were found inside or outside the endoneurial tube labeled with antibody against laminin (LAM). (D) Three weeks (3W) after injury, remyelinating Schwann cells (labeled with MBP or LAM) were devoid of LAMP1 staining, whereas many LAMP1-positive macrophages were still present around remyelinating Schwann cells. DIC, differential interference contrast. Scale bars=100 µm.

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  Fig. 2 Lysosomal acidification is required for demyelination. (A) Differential interference microscopy of teased nerve fibers after 3 days of culture in vitro (3DIV). Scale bar=200 µm. (B) Quantitative result showing myelin ovoid index. (C, D) Western blot analysis showing the levels of myelin basic protein (MBP) (C) and lysosomal associated membrane protein 1 (LAMP1) (D) in cultured nerve explants. "C" indicates uninjured sciatic nerves. CQ, chloroquine; BFA, bafilomycin A.

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  Fig. 3 Lysosomal acidification inhibitors prevent p75 nerve growth factor receptor (p75NGFR) induction. (A) Immunofluorescence microscopy showing p75NGFR expression in teased nerve fibers. Neurofilament (NF) staining showed extensive fragmentation at 3 days culture in vitro (3DIV), regardless of the inhibition of lysosomal acidification. (B) Western blot analysis showing the levels of p75NGFR in cultured nerve explants at 3DIV. (C) Reverse transcription-polymerase chain reaction experiment showing the induction of p75NGFR mRNA in cultured nerve explants at 3 days. (D) Western blot analysis showing the levels of p75NGFR in the sciatic nerves of tumor necrosis factor-α null mice (TNF-KO). (E) Immunofluorescence microscopy showing p75NGFR and lysosomal associated membrane protein 1 (LAMP1) expression in teased nerve fibers 3 days (3d) after injury. "C" indicates uninjured sciatic nerves. BFA, bafilomycin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 3d, three days after crush; WT, wild-type; Con, control. Scale bars=100 µm.

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