A role for glia maturation factor dependent activation of mast cells and microglia in MPTP induced dopamine loss and behavioural deficits in mice

Abstract

The molecular mechanism mediating degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) is not yet fully understood. Previously, we have shown the contribution of glia maturation factor (GMF), a proinflammatory protein in dopaminergic neurodegeneration mediated by activation of mast cells (MCs). In this study, methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal neurodegeneration and astro-glial activations were determined by western blot and immunofluorescence techniques in wild type (WT) mice, MC-deficient (MC-KO) mice and GMF-deficient (GMF-KO) mice, with or without MC reconstitution before MPTP administration. We show that GMF-KO in the MCs reduces the synergistic effects of MC and Calpain1 (calcium-activated cysteine protease enzyme)-dependent dopaminergic neuronal loss that reduces motor behavioral impairments in MPTP-treated mouse. Administration of MPTP increase in calpain-mediated proteolysis in nigral dopaminergic neurons further resulting in motor decline in mice. We found that MPTP administered WT mice exhibits oxidative stress due to significant increases in the levels of malondialdehyde, superoxide dismutase and reduction in the levels of reduced glutathione and glutathione peroxidase activity as compared with both MC-KO and GMF-KO mice. The number of TH-positive neurons in the ventral tegmental area, substantia nigra and the fibers in the striatum were significantly reduced while granulocyte macrophage colony-stimulating factor (GM-CSF), MC-Tryptase, GFAP, IBA1, Calpain1 and intracellular adhesion molecule 1 expression were significantly increased in WT mice. Similarly, tyrosine hydroxylase, dopamine transporters and vesicular monoamine transporters 2 proteins expression were significantly reduced in the SN of MPTP treated WT mice. The motor behavior as analyzed by rotarod and hang test was significantly reduced in WT mice as compared with both the MC-KO and GMF-KO mice. We conclude that GMF-dependent MC activation enhances the detrimental effect of astro-glial activation-mediated oxidative stress and neuroinflammation in the midbrain, and its inhibition may slowdown the progression of PD.

Keywords: Astro-glial activation; Dopaminergic neurodegeneration; Glia maturation factor; Mast cells; Parkinson’s disease.

Fig. 1.

MPTP-induced motor behavioral impairments were assessed by rotarod and hang test performances. MPTP (18 mg/kg by i.p) was administered with and without reconstitution of MC from WT, MC-KO and GMF-KO mice. Mice reconstituted with MC from WT mice shows significant motor behavioral impairments in rotarod by reducing retention time at different RPM (A:5 RPM, B: 10 RPM, C: 15 RPM and D:20 RPM) and hanging time in hang test (E) compared with saline treated control mice, and these impairments were significantly reduced in MC-KO and GMF-KO mice treated with MPTP. Values are presented as mean ± SEM (n = 4). ^∗p < 0.05 saline treated control mice vs MPTP only treated mice. ^ϕp < 0.05 MPTP only treated mice vs WT and GMF-KO MCs reconstituted mice. ^#p < 0.05 wt MC reconstituted mice vs GMF-KO MC reconstituted mice.

Fig. 2.

MPTP induced oxidative stress was determined by analyzing the levels of TBARS (MDA), GSH, activities of SOD and GPx in the SN of the midbrain. Acute neurotoxicity of MPTP (18 mg/kg by i.p) causes significant oxidative stress in WT mice as seen by increasing levels of MDA (A) and the activities of SOD (B), reductions in the levels of GSH (C) and the activities of GPx (D) when compared with control mice. Deficiency of GMF and MC significantly improves these oxidative stress markers expressions when compared with MPTP treated WT mice. Values are presented as mean ± SEM (n = 4). ∗p < 0.05 saline treated control mice vs MPTP treated mice. ^#p < 0.05 wt MC reconstituted mice vs GMF-KO MC reconstituted mice. MDA levels expressed as µmol/mg of protein; GSH level expressed as ng/mg of protein; SOD expressed as units/mg of protein; GPx units-micrograms of glutathione consumed/min.

Fig. 3.

MPTP induce to attenuate TH, and increases GM-CSF and MC-Tryptase expressions in SN of midbrain. Acute administration of MPTP (18 mg/kg) after reconstitution with WT MC significantly reduced TH-protein expression, increased GM-CSF and MC-Tryptase expression in SN of the midbrain as compared with saline control mice as determined by western blot (A) and immunofluorescence (A-L). Mice reconstituted with MC from GMF-KO mice show significant higher TH-protein and reduced GM-CSF and MC-Tryptase expression in dopaminergic neurons in SN of the midbrain. Bar graphs show the effect of MPTP exposure on the GM-CSF and MC-Tryptase expression as compared to the controls (B and C). Values are presented as mean ± SEM (n = 4). ^∗p < 0.05 saline treated control mice vs MPTP treated mice. ^#p < 0.05 wt MC reconstituted mice vs GMF-KO MC reconstituted mice. Scale bar 100 µm.

Fig. 4.

MPTP induced attenuation of TH, DAT and VMAT2 expressions in SN of midbrain. Acute administration of MPTP (18 mg/kg) and reconstitution with WT MC significantly reduced TH, DAT and VMAT2 protein expression when compared with saline control mice as determined by western blot (A). Mice with MC reconstituted from GMF-KO mice and MPTP treatment shows significantly higher expressions of TH, DAT and VMAT2 proteins when compared with reconstitution with WT MC and MPTP treatment. Bar graphs show the effect of MPTP administration on the relative intensity to the control and shows the densitometry of the bands (B, C and D). Values are presented as mean ± SEM (n = 4). ∗p < 0.05 saline treated control mice vs MPTP treated mice. ^#p < 0.05 wt MC reconstituted mice vs GMF-KO MC reconstituted mice.

Fig. 5.

MPTP induced attenuation of TH protein in SN of midbrain. Acute administration of MPTP (18 mg/kg) after reconstitution with WT MC significantly reduced the number of TH-positive dopaminergic neurons and protein expression in SN of the midbrain as compared with saline control mice as determined by immunofluorescence (A-L). Mice reconstituted with MC from GMF-KO mice show a significant higher number of TH-positive dopaminergic neurons in SN of the midbrain. Bar graphs show the effect of MPTP exposure on the number of TH-positive expressing neurons compared to the controls (M). Values are presented as mean ± SEM (n = 4). ∗p < 0.05 saline treated control mice vs MPTP treated mice. ^#p < 0.05 wt MC reconstituted mice vs GMF-KO MC reconstituted mice. Scale bar 100 µm.

Fig. 6.

MPTP induced attenuation of TH protein in VTA of midbrain. Acute treatment with MPTP (18 mg/kg) following reconstitution with WT MC significantly reduced integrated density of TH protein expression in VTA of the midbrain as compared with saline control mice as determined by immunofluorescence (A-L). In sections from mice reconstituted with MCs from GMF-KO mice show a significantly higher integrated density of TH protein expression in VTA of the midbrain when compared with MC reconstitution from WT. Bar graphs show the effect of MPTP exposure on the integrated density of TH expression in VTA to the control (M). Values are presented as mean ± SEM (n = 4). ^∗p < 0.05 saline treated control mice vs MPTP treated mice. ^#p < 0.05 wt MC reconstituted vs GMF-KO MC reconstituted mice. Scale bar 100 µm.

Fig. 7.

MPTP induced attenuation of TH protein in STR of midbrain was determined by immunofluorescence. Acute treatment with MPTP (18 mg/kg) and reconstitution with WT MC significantly reduced integrated density of TH protein expression in STR of the midbrain as compared with control mice. MC reconstitution from GMF-KO mice show a significant higher integrated density of TH protein expression in STR of the midbrain. Bar graphs show the effect of MPTP exposure on the integrated density of TH expression in STR to the control. Values are presented as mean ± SEM (n = 4). ^∗p < 0.05 saline treated control mice vs MPTP treated mice. ^#p < 0.05 wt MC reconstituted mice vs GMF-KO MC reconstituted mice. Scale bar 100 µm.

Fig. 8.

MPTP induced calpain 1 and ICAM 1 expression in SN of the midbrain. Administration of MPTP (18 mg/kg) and WT MC reconstitution significantly increased calpain 1 and ICAM 1 expression in SN of WT mice when compared with saline-treated control mice as determined by western blotting (A). Mice reconstituted with MC from GMF-KO mice followed by MPTP treatment show a significant reduction in calpain 1 and ICAM 1 protein expressions when compared with those from WT MC reconstitution. Bar graphs show the effect of MPTP administration on the relative intensity to the control and shows the densitometry of the bands (B and C). Values are presented as mean ± SEM (n = 4). ^∗p < 0.05 saline treated control mice vs MPTP treated mice. ^#p < 0.05 wt MC reconstituted mice vs GMF-KO MC reconstituted mice.

Fig. 9.

MPTP decreases TH and enhances calpain 1 expression in dopaminergic neurons of SN of the midbrain. Administration of MPTP (18 mg/kg) following MC reconstituted from WT qualitatively increased TH (green fluorescent) and calpain 1 (red fluorescent) expression when compared with saline control mice, GMF-KO MC reconstituted mice show qualitatively lesser expression of these proteins as compared with WT MC reconstituted mice. Representative images: A, E and I saline treated control mice; B, F and J MPTP (18 mg/kg) only treated mice; C, G and K reconstituted with WT MC and MPTP treated mice; D, H and L reconstituted with GMF-KO MC and MPTP treated mice. Scale bar 25 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 10.

MPTP enhances IBA1 and calpain 1 expression in the SN of the midbrain. Administration of MPTP (18 mg/kg) after reconstitution with MC from WT mice qualitatively increased IBA1 (red fluorescence) and calpain 1 (green fluorescence) expression when compared with saline control mice, With mice reconstituted with GMF-KO MC qualitatively lesser expression of these proteins was seen when compared with WT MC reconstituted mice. Bar graphs shows the total positive area (M) and total average intensity (N) of GFAP and ICAM 1 expressions were quantitatively increased in mice reconstituted with MC from WT. Representative images: A, E and I saline treated control mice; B, F and J MPTP (18 mg/kg) only treated mice; C, G and K reconstituted with WT MC and MPTP treated mice; D, H and L reconstituted with GMF-KO MC and MPTP treated mice. Scale bar 25 µm; au arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 11.

MPTP enhances IBA1 and ICAM 1 expression in SN of the midbrain. Administration of MPTP (18 mg/kg) after MC reconstituted from WT mice qualitatively increased IBA1 (red fluorescence) and ICAM 1 (green fluorescence) expression when compared with saline control mice, Mice reconstituted with GMF-KO MC show qualitatively lesser expression of these proteins as compared with WT MC reconstituted mice. Bar graphs shows the total positive area (M) and total average intensity (N) of IBA1 and ICAM 1 expressions were quantitatively increased in mice reconstituted with MC from WT. Representative images: A, E and I saline treated control mice; B, F and J MPTP (18 mg/kg) only treated mice; C, G and K reconstituted with WT MC and MPTP treated mice; D, H and L reconstituted with GMF-KO MC and MPTP treated mice. Scale bar 25 µm; au arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 12.

MPTP enhances GFAP and ICAM 1 expression in SN of the midbrain. Administration of MPTP (18 mg/kg) to mice reconstituted with MC from WT qualitatively increased GFAP (green fluorescence) and ICAM 1 (red fluorescence) expression when compared with saline control mice, mice reconstituted with GMF-KO MC show qualitatively lesser expression of these proteins as compared with WT MC reconstituted mice. Bar graphs shows the total positive area (M) and total average intensity (N) of GFAP and ICAM 1 expressions were quantitatively increased in mice reconstituted with MC from WT. Representative images: A, E and I saline treated control mice; B, F and J MPTP (18 mg/kg) only treated mice; C, G and K reconstituted with WT MC and MPTP treated mice; D, H and L reconstituted with GMF-KO MC and MPTP treated mice. Scale bar 25 µm; au arbitrary units. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Schematic diagram 1.

Mice were acclimatized in the procedure room for the first 3 days (1–3). Behavioral training was started on the 4th day and continued until the 8th day. After a day, animals were injected with MC (i.v), which were obtained from the bone marrow of WT or GMF-KO mice. After 3 days the animals were injected with MPTP (18 mg/kg, i,p) for a total of four injections at 2 h intervals in a day. Behavioral studies were performed after 7 days following the last MPTP injection on the 20th day of our experimental period. After this, the mice were sacrificed and brain tissues were acquired for the biochemical and protein expression studies.