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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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Neuron-specific expression of human GARS proteins in the spinal cord. (A) Schematic representation of the promoter and expression units for each adenovirus (GARS WT, L129P, and G240R); the mitochondrial targeting sequence was deleted to protect GARS proteins from expression in mitochondria. (B) Neuron-specific expression of WT GARS (hGARS) proteins in the spinal cord. GFP expression (green) and immunofluorescence labeling of FLAG (red) in spinal cord cross-sections. Yellow staining indicates an overlap of green and red labeling. GFP/FLAG double positive signals indicate the expression of FLAG-tagged hGARS fusion proteins in the spinal cord. Empt, empty virus without insert; WT, wild-type; L129P, L129P mutant hGARS; G240R, G240R mutant hGARS. Scale bar = 500 µm.
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Figure 1: Neuron-specific expression of human GARS proteins in the spinal cord. (A) Schematic representation of the promoter and expression units for each adenovirus (GARS WT, L129P, and G240R); the mitochondrial targeting sequence was deleted to protect GARS proteins from expression in mitochondria. (B) Neuron-specific expression of WT GARS (hGARS) proteins in the spinal cord. GFP expression (green) and immunofluorescence labeling of FLAG (red) in spinal cord cross-sections. Yellow staining indicates an overlap of green and red labeling. GFP/FLAG double positive signals indicate the expression of FLAG-tagged hGARS fusion proteins in the spinal cord. Empt, empty virus without insert; WT, wild-type; L129P, L129P mutant hGARS; G240R, G240R mutant hGARS. Scale bar = 500 µm.

Mentions: The mouse choline acetyltransferase (ChAT) gene (GenBank accession number NC_000080) was used as the promoter, and included the neuron-restrictive silencer element (NRSE) and a cholinergic-specific enhancer, which are key regulatory elements for neuron-specific expression (18, 19). The use of human GARS (hGARS) clones allowed for subcloning into the pAdTrack-ChAT-CMV/GFP vector. The E1/E3 double-deleted supercoiled adenoviral backbone vector (pAdEasy-1; Stratagene; Santa Clara, CA, USA) was used to generate stable homologous recombinants with pAdTrack-ChAT-hGARSwt-CMV/GFP, pAdTrack-ChAT-hGARSL129P-CMV/GFP, and pAdTrack-ChAT-hGARSL129P-CMV/GFP (Fig. 1A). The titers of Ad/ChAT, AdhGARSwt/ChAT, AdhGARSG230R/ChAT, and AdhGARSL129P/ChAT employed in these experiments were 3.2×109, 5.1×109, 4.9×109, and 2.6×109 pfu/mL, respectively.


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)

Neuron-specific expression of human GARS proteins in the spinal cord. (A) Schematic representation of the promoter and expression units for each adenovirus (GARS WT, L129P, and G240R); the mitochondrial targeting sequence was deleted to protect GARS proteins from expression in mitochondria. (B) Neuron-specific expression of WT GARS (hGARS) proteins in the spinal cord. GFP expression (green) and immunofluorescence labeling of FLAG (red) in spinal cord cross-sections. Yellow staining indicates an overlap of green and red labeling. GFP/FLAG double positive signals indicate the expression of FLAG-tagged hGARS fusion proteins in the spinal cord. Empt, empty virus without insert; WT, wild-type; L129P, L129P mutant hGARS; G240R, G240R mutant hGARS. Scale bar = 500 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4129208&req=5

Figure 1: Neuron-specific expression of human GARS proteins in the spinal cord. (A) Schematic representation of the promoter and expression units for each adenovirus (GARS WT, L129P, and G240R); the mitochondrial targeting sequence was deleted to protect GARS proteins from expression in mitochondria. (B) Neuron-specific expression of WT GARS (hGARS) proteins in the spinal cord. GFP expression (green) and immunofluorescence labeling of FLAG (red) in spinal cord cross-sections. Yellow staining indicates an overlap of green and red labeling. GFP/FLAG double positive signals indicate the expression of FLAG-tagged hGARS fusion proteins in the spinal cord. Empt, empty virus without insert; WT, wild-type; L129P, L129P mutant hGARS; G240R, G240R mutant hGARS. Scale bar = 500 µm.
Mentions: The mouse choline acetyltransferase (ChAT) gene (GenBank accession number NC_000080) was used as the promoter, and included the neuron-restrictive silencer element (NRSE) and a cholinergic-specific enhancer, which are key regulatory elements for neuron-specific expression (18, 19). The use of human GARS (hGARS) clones allowed for subcloning into the pAdTrack-ChAT-CMV/GFP vector. The E1/E3 double-deleted supercoiled adenoviral backbone vector (pAdEasy-1; Stratagene; Santa Clara, CA, USA) was used to generate stable homologous recombinants with pAdTrack-ChAT-hGARSwt-CMV/GFP, pAdTrack-ChAT-hGARSL129P-CMV/GFP, and pAdTrack-ChAT-hGARSL129P-CMV/GFP (Fig. 1A). The titers of Ad/ChAT, AdhGARSwt/ChAT, AdhGARSG230R/ChAT, and AdhGARSL129P/ChAT employed in these experiments were 3.2×109, 5.1×109, 4.9×109, and 2.6×109 pfu/mL, respectively.

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