J Korean Med Sci.  2014 Aug;29(8):1138-1144. 10.3346/jkms.2014.29.8.1138.

Neuropathic Pain Model of Peripheral Neuropathies Mediated by Mutations of Glycyl-tRNA Synthetase

• Lee SJ
• Seo AJ
• Park BS
• Jo HW
• Huh Y

• KMID: 2129611
• DOI: http://doi.org/10.3346/jkms.2014.29.8.1138

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.

Keyword

Charcot-Marie-Tooth Disease; Neuralgia; Glycyl-tRNA Ligase; ATF3 Protein Mouse; Microglia; Adenoviridae

MeSH Terms

Animals
Charcot-Marie-Tooth Disease/*diagnosis/*physiopathology
*Disease Models, Animal
Glycine-tRNA Ligase/*genetics/metabolism
Male
Mice
Mice, Inbred C57BL
Mice, Transgenic
Mutagenesis, Site-Directed
Mutation/genetics
Neuralgia/*diagnosis/*physiopathology
Glycine-tRNA Ligase

Figure

• 

  Fig. 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.

• 

  Fig. 2 Increased number of microglia in the GARS-mutant-expressed dorsal horn. (A) Increase in the number of Iba1-positive microglia in the dorsal horn of the L5-spinal cord 7 days after the transfection of adenovirus vectors into sciatic nerves. GFP expression (green) and immunofluorescence labeling of Iba1 (red) were identified in spinal cord cross-sections. Lower panels are higher magnification images of the spinal dorsal horn. Middle two panels indicates the high magnification images of the upper panels (arrows). Scale bar = 500 µm. (B) Quantification of activated microglia in the dorsal horn at Day 7. The number of Iba1 was calculated with the correction using GFP-neuronal bodies in spinal cord. *P < 0.001 compared with GARS WT (WT, n = 5; L129P, n = 5; G240R, n = 4). (C) mRNA expression of FLAG-tag in the spinal cord following infection with GARS-WT-expressing adenoviruses by RT-PCR.

• 

  Fig. 3 GARS mutant proteins induce ERK activation in the spinal cord. (A) Western blotting revealed an increase in both pERK1 (p44 MAPK) and pERK2 (p42 MAPK) in the L5-spinal cord. FLAG was the adenovirus vector-expressing control. (B) Quantification of pERK1 and pERK2 levels in the L5-spinal cord; pERK1/2 levels were normalized to FLAG levels. *P < 0.001 compared to the WT, n = 4. (C) GARS mutants L129P and G240R induced a significant decrease in paw withdrawal threshold in virus-infected mice compared with Empt- and GATS-WT-expressing mice. Empt, empty virus without insert. n = 4.

• 

  Fig. 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.

Reference

1. Baron R. Mechanisms of disease: neuropathic pain: a clinical perspective. Nat Clin Pract Neurol. 2006; 2:95–106.

2. Woolf CJ, Salter MW. Neuronal plasticity: increasing the gain in pain. Science. 2000; 288:1765–1769.

3. Costigan M, Scholz J, Woolf CJ. Neuropathic pain: a maladaptive response of the nervous system to damage. Annu Rev Neurosci. 2009; 32:1–32.

4. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature. 2003; 424:778–783.

5. Tsuda M, Inoue K, Salter MW. Neuropathic pain and spinal microglia: a big problem from molecules in "small" glia. Trends Neurosci. 2005; 28:101–107.

6. Emery AE. Population frequencies of inherited neuromuscular diseases: a world survey. Neuromuscul Disord. 1991; 1:19–29.

7. Carter GT, Jensen MP, Galer BS, Kraft GH, Crabtree LD, Beardsley RM, Abresch RT, Bird TD. Neuropathic pain in Charcot-Marie-Tooth disease. Arch Phys Med Rehabil. 1998; 79:1560–1564.

8. Pazzaglia C, Vollono C, Ferraro D, Virdis D, Lupi V, Le Pera D, Tonali P, Padua L, Valeriani M. Mechanisms of neuropathic pain in patients with Charcot-Marie-Tooth 1 A: a laser-evoked potential study. Pain. 2010; 149:379–385.

9. Ribiere C, Bernardin M, Sacconi S, Delmont E, Fournier-Mehouas M, Rauscent H, Benchortane M, Staccini P, Lantéri-Minet M, Desnuelle C. Pain assessment in Charcot-Marie-Tooth (CMT) disease. Ann Phys Rehabil Med. 2012; 55:160–173.

10. Antonellis A, Ellsworth RE, Sambuughin N, Puls I, Abel A, Lee-Lin SQ, Jordanova A, Kremensky I, Christodoulou K, Middleton LT, et al. Glycyl tRNA synthetase mutations in Charcot-Marie-Tooth disease type 2D and distal spinal muscular atrophy type V. Am J Hum Genet. 2003; 72:1293–1299.

11. Sambuughin N, Sivakumar K, Selenge B, Lee HS, Friedlich D, Baasanjav D, Dalakas MC, Goldfarb LG. Autosomal dominant distal spinal muscular atrophy type V (dSMA-V) and Charcot-Marie-Tooth disease type 2D (CMT2D) segregate within a single large kindred and map to a refined region on chromosome 7p15. J Neurol Sci. 1998; 161:23–28.

12. Ionasescu V, Searby C, Sheffield VC, Roklina T, Nishimura D, Ionasescu R. Autosomal dominant Charcot-Marie-Tooth axonal neuropathy mapped on chromosome 7p (CMT2D). Hum Mol Genet. 1996; 5:1373–1375.

13. Motley WW, Talbot K, Fischbeck KH. GARS axonopathy: not every neuron's cup of tRNA. Trends Neurosci. 2010; 33:59–66.

14. Bráz JM, Basbaum AI. Differential ATF3 expression in dorsal root ganglion neurons reveals the profile of primary afferents engaged by diverse noxious chemical stimuli. Pain. 2010; 150:290–301.

15. Ivanavicius SP, Ball AD, Heapy CG, Westwood FR, Murray F, Read SJ. Structural pathology in a rodent model of osteoarthritis is associated with neuropathic pain: increased expression of ATF-3 and pharmacological characterisation. Pain. 2007; 128:272–282.

16. Ma W, Quirion R. Partial sciatic nerve ligation induces increase in the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus. Pain. 2002; 99:175–184.

17. Maeda M, Tsuda M, Tozaki-Saitoh H, Inoue K, Kiyama H. Nerve injury-activated microglia engulf myelinated axons in a P2Y12 signaling-dependent manner in the dorsal horn. Glia. 2010; 58:1838–1846.

18. Misawa H, Ishii K, Deguchi T. Gene expression of mouse choline acetyltransferase. Alternative splicing and identification of a highly active promoter region. J Biol Chem. 1992; 267:20392–20399.

19. Lönnerberg P, Schoenherr CJ, Anderson DJ, Ibáñez CF. Cell type-specific regulation of choline acetyltransferase gene expression: role of the neuron-restrictive silencer element and cholinergic-specific enhancer sequences. J Biol Chem. 1996; 271:33358–33365.

20. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods. 1994; 53:55–63.

21. Christodoulou K, Kyriakides T, Hristova AH, Georgiou DM, Kalaydjieva L, Yshpekova B, Ivanova T, Weber JL, Middleton LT. Mapping of a distal form of spinal muscular atrophy with upper limb predominance to chromosome 7p. Hum Mol Genet. 1995; 4:1629–1632.

22. Marchand F, Perretti M, McMahon SB. Role of the immune system in chronic pain. Nat Rev Neurosci. 2005; 6:521–532.

23. Watkins LR, Maier SF. Glia: a novel drug discovery target for clinical pain. Nat Rev Drug Discov. 2003; 2:973–985.

24. Zhuang ZY, Gerner P, Woolf CJ, Ji RR. ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain. 2005; 114:149–159.

25. Ma W, Quirion R. Partial sciatic nerve ligation induces increase in the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus. Pain. 2002; 99:175–184.

26. Tsujino H, Kondo E, Fukuoka T, Dai Y, Tokunaga A, Miki K, Yonenobu K, Ochi T, Noguchi K. Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: a novel neuronal marker of nerve injury. Mol Cell Neurosci. 2000; 15:170–182.

27. Bellier JP, Kimura H. Acetylcholine synthesis by choline acetyltransferase of a peripheral type as demonstrated in adult rat dorsal root ganglion. J Neurochem. 2007; 101:1607–1618.

28. Matsumoto M, Xie W, Inoue M, Ueda H. Evidence for the tonic inhibition of spinal pain by nicotinic cholinergic transmission through primary afferents. Mol Pain. 2007; 3:41.

29. Nangle LA, Zhang W, Xie W, Yang XL, Schimmel P. Charcot-Marie-Tooth disease-associated mutant tRNA synthetases linked to altered dimer interface and neurite distribution defect. Proc Natl Acad Sci U S A. 2007; 104:11239–11244.

30. Seo AJ, Shin YH, Lee SJ, Kim D, Park BS, Kim S, Choi KH, Jeong NY, Park C, Jang JY, et al. A novel adenoviral vector-mediated mouse model of Charcot-Marie-Tooth type 2D (CMT2D). J Mol Histol. 2014; 45:121–128.