Anat Cell Biol.  2017 Mar;50(1):48-59. 10.5115/acb.2017.50.1.48.

Potential involvement of glycogen synthase kinase (GSK)-3β in a rat model of multiple sclerosis: evidenced by lithium treatment

Affiliations
  • 1Department of Veterinary Anatomy, College of Veterinary Medicine, Jeju National University, Jeju, Korea. shint@jejunu.ac.kr
  • 2Department of Molecular Anatomy, School of Medicine, University of the Ryukyus, Nishihara, Japan.
  • 3Eco-friendly Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Korea.
  • 4Department of Veterinary Anatomy, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea.

Abstract

Glycogen synthase kinase (GSK)-3β has been known as a pro-inflammatory molecule in neuroinflammation. The involvement of GSK-3β remains unsolved in acute monophasic rat experimental autoimmune encephalomyelitis (EAE). The aim of this study was to evaluate a potential role of GSK-3β in central nervous system (CNS) autoimmunity through its inhibition by lithium. Lithium treatment significantly delayed the onset of EAE paralysis and ameliorated its severity. Lithium treatment reduced the serum level of pro-inflammatory tumor necrosis factor a but not that of interleukin 10. Western blot analysis showed that the phosphorylation of GSK-3β (p-GSK-3β) and its upstream factor Akt was significantly increased in the lithium-treated group. Immunohistochemical examination revealed that lithium treatment also suppressed the activation of ionized calcium binding protein-1-positive microglial cells and vascular cell adhesion molecule-1 expression in the spinal cords of lithium-treated EAE rats. These results demonstrate that lithium ameliorates clinical symptom of acute monophasic rat EAE, and GSK-3 is a target for the suppression of acute neuroinflammation as far as rat model of human CNS disease is involved.

Keyword

Experimental autoimmune encephalomyelitis; GSK-3 signaling; Lithium; Rat model

MeSH Terms

Animals
Autoimmunity
Blotting, Western
Calcium
Central Nervous System
Central Nervous System Diseases
Encephalomyelitis, Autoimmune, Experimental
Glycogen Synthase Kinase 3
Glycogen Synthase Kinases*
Glycogen Synthase*
Glycogen*
Humans
Interleukin-10
Lithium*
Models, Animal*
Multiple Sclerosis*
Paralysis
Phosphorylation
Rats*
Spinal Cord
Tumor Necrosis Factor-alpha
Vascular Cell Adhesion Molecule-1
Calcium
Glycogen
Glycogen Synthase
Glycogen Synthase Kinase 3
Glycogen Synthase Kinases
Interleukin-10
Lithium
Tumor Necrosis Factor-alpha
Vascular Cell Adhesion Molecule-1

Figure

  • Fig. 1 Western blot analysis of phosphorylated glycogen synthase kinase-3β (p-GSK-3β) and total GSK-3β expression in the spinal cords of rats with experimental autoimmune encephalomyelitis (EAE). (A) Representative Western blot of p-GSK-3β, total GSK-3β, and β-actin expression. (B) Semi-quantitative analysis of p-GSK-3β expression in the spinal cord. p-GSK-3β expression was significantly decreased on day 13 postimmunization (PI) compared with normal controls. Values are presented as means±SE. *P<0.05 vs. controls.

  • Fig. 2 Immunohistochemical staining of phosphorylated glycogen synthase kinase-3β (p-GSK-3β) in the spinal cords of normal (A) and experimental autoimmune encephalomyelitis (EAE) rats on day 13 postimmunization (PI) (G.3) (B–D) and day 21 PI (R.0) (E). p-GSK-3β in astrocytes (arrows) in the spinal cords of normal controls. On day 13 PI, p-GSK-3β was expressed by infiltrating inflammatory cells (B and E, arrowheads), astrocytes (B and E, arrows), neurons (C, arrows), vascular endothelial cells (D, “V”) and ependymal cells (D) in the spinal cord. Primary antibody was omitted (F). Counterstained with hematoxylin. Scale bars=25 µm (A–F).

  • Fig. 3 (A) Clinical signs during lithium treatment of experimental autoimmune encephalomyelitis (EAE). Animals (n=5) were euthanized at the time of peak paralysis (day 13 postimmunization [PI], arrow). (B–D) Histopathological examination of the spinal cords of rats with EAE on day 13 PI. The spinal cords of the control group showed a normal architecture (B). However, the spinal cords of vehicle-treated rats contained many inflammatory cells in the parenchyma (C), whereas there were fewer inflammatory cells in the spinal cords of lithium-treated rats (D). (B–D) Hematoxylin and eosin staining. Values are presented as mean±SE. **P< 0.01 vs. vehicle treatment. Scale bars=50 µm (B–D).

  • Fig. 4 Immunohistochemical localization of ionized calcium-binding protein-1 (Iba-1) in the spinal cords of normal control, vehicle-treated, and lithium-treated rats (A). Semi-quantitative analysis of Iba-1 immunoreactivity in normal control and vehicle- and lithium-treated rats with experimental autoimmune encephalomyelitis (EAE) on day 13 postimmunization (PI) (B). Counterstained with hematoxylin. Values are presented as mean±SE. **P<0.01 vs. normal controls (n=5 per group), ##P<0.01 vs. vehicle treatment. Scale bars=200 µm (A).

  • Fig. 5 Serum tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) levels. Sera were collected on day 13 postimmunization (PI) and TNF-α (A) and IL-10 (B) levels were measured by enzyme-linked immunosorbent assay. EAE, experimental autoimmune encephalomyelitis. Values are presented as mean±SE. *P<0.05 vs. normal controls (n=5 per group), #P<0.05 vs. vehicle treatment.

  • Fig. 6 Western blot analysis of the effect of lithium treatment on glycogen synthase kinase-3 (GSK-3) activity in the spinal cords of experimental autoimmune encephalomyelitis (EAE) rats. (A) Representative immunoblots of p-Akt (Ser473), total Akt (~51 kDa), p-GSK-3β (Ser9), total GSK-3β (~46 kDa), β-catenin (~92 kDa), and β-actin (~45 kDa). (B, C) Bar graphs show significant decreases in p-Akt, p-GSK-3β, and β-catenin expression in the spinal cords of vehicle-treated rats. Lithium treatment markedly increased the expression levels of these factors. To quantify phosphorylation of Akt and GSK-3β, levels of the phosphorylated forms were normalized to total Akt or GSK-3β. For normalization of β-catenin expression, the membranes were reprobed using an anti–β-actin antibody. Values are mean±SE. n = 6 per group. *P<0.05 vs. controls, #P<0.05 vs. lithium treatment. N, normal controls; V, vehicle-treated EAE; Li, lithium-treated EAE.

  • Fig. 7 Immunohistochemical detection of vascular cell adhesion molecule 1 (VCAM-1) in the spinal cords of normal control (A), vehicle-treated (B), and lithium-treated (C) rats. VCAM-1 in vascular endothelial cells (asterisk) and astrocytes (arrow) of the spinal cords of normal control rats (A). VCAM-1 immunoreactivity was detected mainly in the vascular endothelial cells (asterisk), infiltrated inflammatory cells, and astrocytes (arrows) in vehicle-treated rats (B), but it was rarely detected in the spinal cords of lithium-treated rats (C). Semi-quantitative analysis of VCAM-1 immunoreactivity in normal control and vehicle- and lithium-treated rats with experimental autoimmune encephalomyelitis (EAE) on day 13 postimmunization (PI) (D). Counterstained with hematoxylin. Values are presented as mean±SE. *P<0.05 vs. normal controls (n=5 each group), #P<0.05 vs. vehicle treatment. Scale bars=25 µm (A–C).


Reference

1. Shin T, Ahn M, Matsumoto Y. Mechanism of experimental autoimmune encephalomyelitis in Lewis rats: recent insights from macrophages. Anat Cell Biol. 2012; 45:141–148. PMID: 23094201.
2. Shin T, Kojima T, Tanuma N, Ishihara Y, Matsumoto Y. The subarachnoid space as a site for precursor T cell proliferation and effector T cell selection in experimental autoimmune encephalomyelitis. J Neuroimmunol. 1995; 56:171–178. PMID: 7860712.
3. Swanborg RH. Experimental autoimmune encephalomyelitis in the rat: lessons in T-cell immunology and autoreactivity. Immunol Rev. 2001; 184:129–135. PMID: 12086308.
4. Matsumoto Y. New approach to immunotherapy against organ-specific autoimmune diseases with T cell receptor and chemokine receptor DNA vaccines. Curr Drug Targets Immune Endocr Metabol Disord. 2005; 5:73–77. PMID: 15777206.
5. Ahn M, Yang W, Kim H, Jin JK, Moon C, Shin T. Immunohistochemical study of arginase-1 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis. Brain Res. 2012; 1453:77–86. PMID: 22483960.
6. Ellrichmann G, Thöne J, Lee DH, Rupec RA, Gold R, Linker RA. Constitutive activity of NF-kappa B in myeloid cells drives pathogenicity of monocytes and macrophages during autoimmune neuroinflammation. J Neuroinflammation. 2012; 9:15. PMID: 22260436.
7. Kim H, Moon C, Ahn M, Lee Y, Kim S, Matsumoto Y, Koh CS, Kim MD, Shin T. Increased phosphorylation of cyclic AMP response element-binding protein in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis. Brain Res. 2007; 1162:113–120. PMID: 17617386.
8. Aleshin S, Strokin M, Sergeeva M, Reiser G. Peroxisome proliferator-activated receptor (PPAR)beta/delta, a possible nexus of PPARalpha- and PPARgamma-dependent molecular pathways in neurodegenerative diseases: Review and novel hypotheses. Neurochem Int. 2013; 63:322–330. PMID: 23811400.
9. Chen G, Shannon M. Transcription factors and th17 cell development in experimental autoimmune encephalomyelitis. Crit Rev Immunol. 2013; 33:165–182. PMID: 23582061.
10. Beurel E. Regulation by glycogen synthase kinase-3 of inflammation and T cells in CNS diseases. Front Mol Neurosci. 2011; 4:18. PMID: 21941466.
11. Beurel E, Kaidanovich-Beilin O, Yeh WI, Song L, Palomo V, Michalek SM, Woodgett JR, Harrington LE, Eldar-Finkelman H, Martinez A, Jope RS. Regulation of Th1 cells and experimental autoimmune encephalomyelitis by glycogen synthase kinase-3. J Immunol. 2013; 190:5000–5011. PMID: 23606540.
12. Rowse AL, Naves R, Cashman KS, McGuire DJ, Mbana T, Raman C, De Sarno P. Lithium controls central nervous system autoimmunity through modulation of IFN-gamma signaling. PLoS One. 2012; 7:e52658. PMID: 23285134.
13. De Sarno P, Axtell RC, Raman C, Roth KA, Alessi DR, Jope RS. Lithium prevents and ameliorates experimental autoimmune encephalomyelitis. J Immunol. 2008; 181:338–345. PMID: 18566399.
14. Grimes CA, Jope RS. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog Neurobiol. 2001; 65:391–426. PMID: 11527574.
15. Eto M, Kouroedov A, Cosentino F, Lüscher TF. Glycogen synthase kinase-3 mediates endothelial cell activation by tumor necrosis factor-alpha. Circulation. 2005; 112:1316–1322. PMID: 16129813.
16. Ramirez SH, Fan S, Zhang M, Papugani A, Reichenbach N, Dykstra H, Mercer AJ, Tuma RF, Persidsky Y. Inhibition of glycogen synthase kinase 3beta (GSK3beta) decreases inflammatory responses in brain endothelial cells. Am J Pathol. 2010; 176:881–892. PMID: 20056834.
17. Lee MJ, Jang M, Choi J, Chang BS, Kim DY, Kim SH, Kwak YS, Oh S, Lee JH, Chang BJ, Nah SY, Cho IH. Korean red ginseng and ginsenoside-Rb1/-Rg1 alleviate experimental autoimmune encephalomyelitis by suppressing Th1 and Th17 cells and upregulating regulatory T cells. Mol Neurobiol. 2016; 53:1977–2002. PMID: 25846819.
18. Jope RS. Lithium and GSK-3: one inhibitor, two inhibitory actions, multiple outcomes. Trends Pharmacol Sci. 2003; 24:441–443. PMID: 12967765.
19. Levine S, Saltzman A. Inhibition of experimental allergic encephalomyelitis by lithium chloride: specific effect or nonspecific stress? Immunopharmacology. 1991; 22:207–213. PMID: 1663498.
20. Kim S, Moon C, Wie MB, Kim H, Tanuma N, Matsumoto Y, Shin T. Enhanced expression of constitutive and inducible forms of nitric oxide synthase in autoimmune encephalomyelitis. J Vet Sci. 2000; 1:11–17. PMID: 14612615.
21. Kim MD, Cho HJ, Shin T. Expression of osteopontin and its ligand, CD44, in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis. J Neuroimmunol. 2004; 151:78–84. PMID: 15145606.
22. De Sarno P, Li X, Jope RS. Regulation of Akt and glycogen synthase kinase-3 beta phosphorylation by sodium valproate and lithium. Neuropharmacology. 2002; 43:1158–1164. PMID: 12504922.
23. Kim H, Moon C, Ahn M, Byun J, Lee Y, Kim MD, Matsumoto Y, Koh CS, Shin T. Heat shock protein 27 upregulation and phosphorylation in rat experimental autoimmune encephalomyelitis. Brain Res. 2009; 1304:155–163. PMID: 19781527.
24. Dill J, Wang H, Zhou F, Li S. Inactivation of glycogen synthase kinase 3 promotes axonal growth and recovery in the CNS. J Neurosci. 2008; 28:8914–8928. PMID: 18768685.
25. Tafreshi AP, Payne N, Sun G, Sylvain A, Schulze K, Bernard C. Inactive GSK3beta is disturbed in the spinal cord during experimental autoimmune encephalomyelitis, but rescued by stem cell therapy. Neuroscience. 2014; 277:498–505. PMID: 25064057.
26. Weng HR, Gao M, Maixner DW. Glycogen synthase kinase 3 beta regulates glial glutamate transporter protein expression in the spinal dorsal horn in rats with neuropathic pain. Exp Neurol. 2014; 252:18–27. PMID: 24275526.
27. Li H, Li Q, Du X, Sun Y, Wang X, Kroemer G, Blomgren K, Zhu C. Lithium-mediated long-term neuroprotection in neonatal rat hypoxia-ischemia is associated with antiinflammatory effects and enhanced proliferation and survival of neural stem/progenitor cells. J Cereb Blood Flow Metab. 2011; 31:2106–2115. PMID: 21587270.
28. Kang K, Kim YJ, Kim YH, Roh JN, Nam JM, Kim PY, Ryu WS, Lee SH, Yoon BW. Lithium pretreatment reduces brain injury after intracerebral hemorrhage in rats. Neurol Res. 2012; 34:447–454. PMID: 22450252.
29. Dong H, Zhang X, Dai X, Lu S, Gui B, Jin W, Zhang S, Zhang S, Qian Y. Lithium ameliorates lipopolysaccharide-induced microglial activation via inhibition of toll-like receptor 4 expression by activating the PI3K/Akt/FoxO1 pathway. J Neuroinflammation. 2014; 11:140. PMID: 25115727.
30. Doverhag C, Hedtjarn M, Poirier F, Mallard C, Hagberg H, Karlsson A, Sävman K. Galectin-3 contributes to neonatal hypoxic-ischemic brain injury. Neurobiol Dis. 2010; 38:36–46. PMID: 20053377.
31. Chanaday NL, Roth GA. Microglia and astrocyte activation in the frontal cortex of rats with experimental autoimmune encephalomyelitis. Neuroscience. 2016; 314:160–169. PMID: 26679600.
32. Chen K, Wu Y, Zhu M, Deng Q, Nie X, Li M, Wu M, Huang X. Lithium chloride promotes host resistance against Pseudomonas aeruginosa keratitis. Mol Vis. 2013; 19:1502–1514. PMID: 23878501.
33. Liu KJ, Lee YL, Yang YY, Shih NY, Ho CC, Wu YC, Huang TS, Huang MC, Liu HC, Shen WW, Leu SJ. Modulation of the development of human monocyte-derived dendritic cells by lithium chloride. J Cell Physiol. 2011; 226:424–433. PMID: 20672290.
34. Valvassori SS, Tonin PT, Varela RB, Carvalho AF, Mariot E, Amboni RT, Bianchini G, Andersen ML, Quevedo J. Lithium modulates the production of peripheral and cerebral cytokines in an animal model of mania induced by dextroamphetamine. Bipolar Disord. 2015; 17:507–517. PMID: 25929806.
35. Maixner DW, Weng HR. The role of glycogen synthase kinase 3 beta in neuroinflammation and pain. J Pharm Pharmacol (Los Angel). 2013; 1:001. PMID: 25309941.
36. Jope RS, Yuskaitis CJ, Beurel E. Glycogen synthase kinase-3 (GSK3): inflammation, diseases, and therapeutics. Neurochem Res. 2007; 32:577–595. PMID: 16944320.
37. Beurel E, Michalek SM, Jope RS. Innate and adaptive immune responses regulated by glycogen synthase kinase-3 (GSK3). Trends Immunol. 2010; 31:24–31. PMID: 19836308.
38. Wu B, Crampton SP, Hughes CC. Wnt signaling induces matrix metalloproteinase expression and regulates T cell transmigration. Immunity. 2007; 26:227–239. PMID: 17306568.
39. Doble BW, Woodgett JR. GSK-3: tricks of the trade for a multitasking kinase. J Cell Sci. 2003; 116(Pt 7):1175–1186. PMID: 12615961.
40. Wang H, Brown J, Martin M. Glycogen synthase kinase 3: a point of convergence for the host inflammatory response. Cytokine. 2011; 53:130–140. PMID: 21095632.
41. Wang G, Shi Y, Jiang X, Leak RK, Hu X, Wu Y, Pu H, Li WW, Tang B, Wang Y, Gao Y, Zheng P, Bennett MV, Chen J. HDAC inhibition prevents white matter injury by modulating microglia/macrophage polarization through the GSK3beta/PTEN/Akt axis. Proc Natl Acad Sci U S A. 2015; 112:2853–2858. PMID: 25691750.
42. Raghavendra PB, Lee E, Parameswaran N. Regulation of macrophage biology by lithium: a new look at an old drug. J Neuroimmune Pharmacol. 2014; 9:277–284. PMID: 24277481.
43. Chen S, Guttridge DC, You Z, Zhang Z, Fribley A, Mayo MW, Kitajewski J, Wang CY. Wnt-1 signaling inhibits apoptosis by activating beta-catenin/T cell factor-mediated transcription. J Cell Biol. 2001; 152:87–96. PMID: 11149923.
44. Shin T, Ahn M, Jung K, Heo S, Kim D, Jee Y, Lim YK, Yeo EJ. Activation of mitogen-activated protein kinases in experimental autoimmune encephalomyelitis. J Neuroimmunol. 2003; 140:118–125. PMID: 12864979.
45. Archambault AS, Sim J, McCandless EE, Klein RS, Russell JH. Region-specific regulation of inflammation and pathogenesis in experimental autoimmune encephalomyelitis. J Neuroimmunol. 2006; 181:122–132. PMID: 17030428.
46. Gimenez MA, Sim JE, Russell JH. TNFR1-dependent VCAM-1 expression by astrocytes exposes the CNS to destructive inflammation. J Neuroimmunol. 2004; 151:116–125. PMID: 15145610.
47. Su Y, Qadri SM, Cayabyab FS, Wu L, Liu L. Regulation of methylglyoxal-elicited leukocyte recruitment by endothelial SGK1/GSK3 signaling. Biochim Biophys Acta. 2014; 1843:2481–2491. PMID: 25003317.
48. Kan QC, Zhu L, Liu N, Zhang GX. Matrine suppresses expression of adhesion molecules and chemokines as a mechanism underlying its therapeutic effect in CNS autoimmunity. Immunol Res. 2013; 56:189–196. PMID: 23549837.
Full Text Links
  • ACB
Actions
Cited
CITED
export Copy
Close
Share
  • Twitter
  • Facebook
Similar articles
Copyright © 2024 by Korean Association of Medical Journal Editors. All rights reserved.     E-mail: koreamed@kamje.or.kr