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

• Ahn M
• Kim J
• Park C
• Cho J
• Jee Y
• Jung K
• Moon C
• Shin T

• KMID: 2390426
• DOI: http://doi.org/10.5115/acb.2017.50.1.48

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).

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