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Neuropathic pain model of peripheral neuropathies mediated by mutations of glycyl-tRNA synthetase.

Lee SJ, Seo AJ, Park BS, Jo HW, Huh Y - J. Korean Med. Sci. (2014)

Bottom Line: To this end, glycyl-tRNA synthetase (GARS) fusion proteins with a FLAG-tag (wild type [WT], L129P and G240R mutants) were expressed in spinal cord and dorsal root ganglion (DRG) neurons using adenovirus vectors.It is known that GARS mutants induce GARS axonopathies, including CMT type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V).These results suggest that this animal model of CMT using an adenovirus may provide information regarding CMT as well as a useful strategy for the treatment of neuropathic pain.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, School of Medicine, Kyung Hee University, Seoul, Korea.

ABSTRACT
Charcot-Marie-Tooth disease (CMT) is the most common inherited motor and sensory neuropathy. Previous studies have found that, according to CMT patients, neuropathic pain is an occasional symptom of CMT. However, neuropathic pain is not considered to be a significant symptom associated with CMT and, as a result, no studies have investigated the pathophysiology underlying neuropathic pain in this disorder. Thus, the first animal model of neuropathic pain was developed by our laboratory using an adenovirus vector system to study neuropathic pain in CMT. To this end, glycyl-tRNA synthetase (GARS) fusion proteins with a FLAG-tag (wild type [WT], L129P and G240R mutants) were expressed in spinal cord and dorsal root ganglion (DRG) neurons using adenovirus vectors. It is known that GARS mutants induce GARS axonopathies, including CMT type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V). Additionally, the morphological phenotypes of neuropathic pain in this animal model of GARS-induced pain were assessed using several possible markers of pain (Iba1, pERK1/2) or a marker of injured neurons (ATF3). These results suggest that this animal model of CMT using an adenovirus may provide information regarding CMT as well as a useful strategy for the treatment of neuropathic pain.

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GARS mutant-induced ATF3 expression in dorsal root ganglion (DRG) neurons. (A) Protein lysates from mouse DRG neurons following infection with adenoviruses were analyzed by Western blotting (WT, wild-type; L129P, L129P mutant hGARS; G240R, G240R mutant hGARS). (B) mRNA expression of FLAG-tag in DRG following infection with adenoviruses by RT-PCR. (C) In GARS mutant-expressed DRG neurons, GARS mutants (L129P and G240R) induced ATF3 expression (red). In contrast, in GARS WT-expressing DRG neurons, identical adenovirus vector expression failed to induce ATF3. Scale bar = 20 µm. (D) Quantification of ATF3 levels in DRG neurons. ATF3 levels were normalized to GFP levels. *P < 0.001 compared to the WT, n = 4.
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Figure 4: GARS mutant-induced ATF3 expression in dorsal root ganglion (DRG) neurons. (A) Protein lysates from mouse DRG neurons following infection with adenoviruses were analyzed by Western blotting (WT, wild-type; L129P, L129P mutant hGARS; G240R, G240R mutant hGARS). (B) mRNA expression of FLAG-tag in DRG following infection with adenoviruses by RT-PCR. (C) In GARS mutant-expressed DRG neurons, GARS mutants (L129P and G240R) induced ATF3 expression (red). In contrast, in GARS WT-expressing DRG neurons, identical adenovirus vector expression failed to induce ATF3. Scale bar = 20 µm. (D) Quantification of ATF3 levels in DRG neurons. ATF3 levels were normalized to GFP levels. *P < 0.001 compared to the WT, n = 4.

Mentions: To identify the pathophysiological mechanisms underlying neuropathic pain, several studies have monitored the induction of ATF3, a selective marker of nerve injury (14, 15, 26). These studies found that stimuli in the peripheral nerves induce the expression of ATF3 in DRG neurons in animal models of neuropathic pain, suggesting that ATF3 has a significant influence on neuropathic pain. Here, ATF3 was also utilized as a possible marker of sensory damage that affects neuropathic pain in mutant GARS-induced neuropathies. A previous report found that immunohistochemically stained ChAT could be visualized in the myelinated A-fibers and unmyelinated C-fibers of the adult rat DRG neurons (27, 28). To identify the expression of GARS fusion proteins in DRG neurons, the expression of the viral protein FLAG was examined in DRG tissues by Western blotting and RT-PCR (Fig. 4A, B). FLAG expression was confirmed in DRG neurons following viral vector transfection into the sciatic nerves (Fig. 4A, B). To assess ATF3 activation in mutant-GARS-expressing DRG neurons compared with WT-GARS-expressing neurons, DRG tissues were immunostained with an anti-ATF3 antibody. ATF3 staining identified an increased amount of mutant-GARS-expressing (L129P and G240R) DRG neurons compared with WT-GARS-expressing neurons (Fig. 4C). ATF3 staining was strong in the area detected by GFP green fluorescence in mutant-GARS-expressing DRG neurons, but no ATF3-positive signals were detected in WT-GARS-expressing DRG neurons (Fig. 4C). Quantitative analyses also revealed an increase in the number of ATF3-positive signals in the area positive for GFP fluorescence in mutant-GARS-expressing DRG neurons compared with WT-GARS-expressing DRG neurons. Thus, these data suggest that mutant GARS protein expression in DRG sensory neurons may be involved in ATF3 activation and that the central axon terminals of DRG neurons associated with ATF3 activation may affect the recruitment of microglia and contribute to the initiation of neuropathic pain in mutant GARS-induced neuropathies.


Neuropathic pain model of peripheral neuropathies mediated by mutations of glycyl-tRNA synthetase.

Lee SJ, Seo AJ, Park BS, Jo HW, Huh Y - J. Korean Med. Sci. (2014)

GARS mutant-induced ATF3 expression in dorsal root ganglion (DRG) neurons. (A) Protein lysates from mouse DRG neurons following infection with adenoviruses were analyzed by Western blotting (WT, wild-type; L129P, L129P mutant hGARS; G240R, G240R mutant hGARS). (B) mRNA expression of FLAG-tag in DRG following infection with adenoviruses by RT-PCR. (C) In GARS mutant-expressed DRG neurons, GARS mutants (L129P and G240R) induced ATF3 expression (red). In contrast, in GARS WT-expressing DRG neurons, identical adenovirus vector expression failed to induce ATF3. Scale bar = 20 µm. (D) Quantification of ATF3 levels in DRG neurons. ATF3 levels were normalized to GFP levels. *P < 0.001 compared to the WT, n = 4.
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Figure 4: GARS mutant-induced ATF3 expression in dorsal root ganglion (DRG) neurons. (A) Protein lysates from mouse DRG neurons following infection with adenoviruses were analyzed by Western blotting (WT, wild-type; L129P, L129P mutant hGARS; G240R, G240R mutant hGARS). (B) mRNA expression of FLAG-tag in DRG following infection with adenoviruses by RT-PCR. (C) In GARS mutant-expressed DRG neurons, GARS mutants (L129P and G240R) induced ATF3 expression (red). In contrast, in GARS WT-expressing DRG neurons, identical adenovirus vector expression failed to induce ATF3. Scale bar = 20 µm. (D) Quantification of ATF3 levels in DRG neurons. ATF3 levels were normalized to GFP levels. *P < 0.001 compared to the WT, n = 4.
Mentions: To identify the pathophysiological mechanisms underlying neuropathic pain, several studies have monitored the induction of ATF3, a selective marker of nerve injury (14, 15, 26). These studies found that stimuli in the peripheral nerves induce the expression of ATF3 in DRG neurons in animal models of neuropathic pain, suggesting that ATF3 has a significant influence on neuropathic pain. Here, ATF3 was also utilized as a possible marker of sensory damage that affects neuropathic pain in mutant GARS-induced neuropathies. A previous report found that immunohistochemically stained ChAT could be visualized in the myelinated A-fibers and unmyelinated C-fibers of the adult rat DRG neurons (27, 28). To identify the expression of GARS fusion proteins in DRG neurons, the expression of the viral protein FLAG was examined in DRG tissues by Western blotting and RT-PCR (Fig. 4A, B). FLAG expression was confirmed in DRG neurons following viral vector transfection into the sciatic nerves (Fig. 4A, B). To assess ATF3 activation in mutant-GARS-expressing DRG neurons compared with WT-GARS-expressing neurons, DRG tissues were immunostained with an anti-ATF3 antibody. ATF3 staining identified an increased amount of mutant-GARS-expressing (L129P and G240R) DRG neurons compared with WT-GARS-expressing neurons (Fig. 4C). ATF3 staining was strong in the area detected by GFP green fluorescence in mutant-GARS-expressing DRG neurons, but no ATF3-positive signals were detected in WT-GARS-expressing DRG neurons (Fig. 4C). Quantitative analyses also revealed an increase in the number of ATF3-positive signals in the area positive for GFP fluorescence in mutant-GARS-expressing DRG neurons compared with WT-GARS-expressing DRG neurons. Thus, these data suggest that mutant GARS protein expression in DRG sensory neurons may be involved in ATF3 activation and that the central axon terminals of DRG neurons associated with ATF3 activation may affect the recruitment of microglia and contribute to the initiation of neuropathic pain in mutant GARS-induced neuropathies.

Bottom Line: To this end, glycyl-tRNA synthetase (GARS) fusion proteins with a FLAG-tag (wild type [WT], L129P and G240R mutants) were expressed in spinal cord and dorsal root ganglion (DRG) neurons using adenovirus vectors.It is known that GARS mutants induce GARS axonopathies, including CMT type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V).These results suggest that this animal model of CMT using an adenovirus may provide information regarding CMT as well as a useful strategy for the treatment of neuropathic pain.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, School of Medicine, Kyung Hee University, Seoul, Korea.

ABSTRACT
Charcot-Marie-Tooth disease (CMT) is the most common inherited motor and sensory neuropathy. Previous studies have found that, according to CMT patients, neuropathic pain is an occasional symptom of CMT. However, neuropathic pain is not considered to be a significant symptom associated with CMT and, as a result, no studies have investigated the pathophysiology underlying neuropathic pain in this disorder. Thus, the first animal model of neuropathic pain was developed by our laboratory using an adenovirus vector system to study neuropathic pain in CMT. To this end, glycyl-tRNA synthetase (GARS) fusion proteins with a FLAG-tag (wild type [WT], L129P and G240R mutants) were expressed in spinal cord and dorsal root ganglion (DRG) neurons using adenovirus vectors. It is known that GARS mutants induce GARS axonopathies, including CMT type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V). Additionally, the morphological phenotypes of neuropathic pain in this animal model of GARS-induced pain were assessed using several possible markers of pain (Iba1, pERK1/2) or a marker of injured neurons (ATF3). These results suggest that this animal model of CMT using an adenovirus may provide information regarding CMT as well as a useful strategy for the treatment of neuropathic pain.

Show MeSH
Related in: MedlinePlus