J Korean Med Sci.  2007 Aug;22(4):641-645. 10.3346/jkms.2007.22.4.641.

Netrin Inhibits Regenerative Axon Growth of Adult Dorsal Root Ganglion Neurons in Vitro

• Park JI
• Seo IA
• Lee HK
• Park HT
• Shin SW
• Park YM
• Ahn KJ

• KMID: 1127079
• DOI: http://doi.org/10.3346/jkms.2007.22.4.641

Abstract

Netrin is a neuronal guidance molecule implicated in the development of spinal commissural neurons and cortical neurons. The attractive function of netrin requires the receptor, Deleted in Colorectal Cancer (DCC), while the receptor Unc5h is involved in the repulsive action of netrin during embryonic development. Although the expression of netrin and its receptor has been demonstrated in the adult nervous system, the function of netrin in adult neurons has not yet been elucidated. Here, we show that netrin treatment inhibited neurite outgrowth of adult dorsal root ganglion (DRG) neurons in explant and dissociated cultures. In addition, unc5h1-3 mRNAs, but not the dcc mRNA, are abundantly expressed in the adult DRG. An in situ hybridization study demonstrated that unc5h mRNAs were expressed in DRG neurons. This finding indicates that netrin/Unc5h signaling may play a role in the neurite outgrowth of adult DRG neurons and that netrin may be involved in the regulation of peripheral nerve regeneration.

Keyword

Netrin; Unc5h; Regeneration; Dorsal Root Ganglion

MeSH Terms

Animals
Axons/*drug effects/physiology
Cells, Cultured
Ganglia, Spinal/cytology/drug effects/metabolism
Gene Expression/drug effects
In Situ Hybridization
Male
Nerve Growth Factors/*pharmacology
Nerve Regeneration/drug effects
Neurites/drug effects/physiology
Neurons/*drug effects/metabolism/physiology
RNA, Messenger/genetics/metabolism
Rats
Rats, Sprague-Dawley
Receptors, Cell Surface/genetics
Reverse Transcriptase Polymerase Chain Reaction
Time Factors
Tissue Culture Techniques
Tumor Suppressor Proteins/*pharmacology

Figure

• 

  Fig. 1 Netrin inhibits the regenerative neurite growth of adult DRG neurons in cultures. (A) Representative images of the control explant (left) and netrin-1 (500 ng/mL) incubated explant (right). (B) Measurement of neurite outgrowth from anti-Tuj1 stained explants. Values shown are the means±s.d. from three independent experiments, and 20-25 explants were counted for each experiment. (C) Representative images of the control (left) and netrin-1 (500 ng/ mL)-treated dissociated neurons (right). (D, E) Measurement of neurite-bearing neurons (D) and length of the longest neurite from 180-200 dissociated neurons for each measurement (E). Values shown are the means±s.d. from three independent experiments (*p<0.05, Student's t-test). Bars in A and C, 100 µm.

• 

  Fig. 2 The expression of netrin receptor mRNAs in the adult DRG Dcc and unc5h1-4 mRNA levels in the adult DRG and spinal cord were evaluated using RT-PCR analysis, and PCR amplification of GAPDH was used as a control. unc5h1-3 mRNAs were expressed in the adult DRG, whereas dcc and unc5h4 mRNAs were not. The values in the right column shows the signal intensity normalized on the basis of the PCR amplification of GAPDH. The values indicate the low levels of unc5h mRNA expression in the DRG as compared to those in the spinal cord. SC; spinal cord, DRG; dorsal root ganglion.

• 

  Fig. 3 unc5h mRNA expression in the adult DRG. The mRNA expressions of unc5h1 and 2 genes were examined by in situ hybridization histochemistry. The figures are the representative images of the staining. unc5h1 and unc5h2 expression was distinct in DRG sensory neurons. Bar, 50 µm. DRG; dorsal root ganglion.

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