The CD200R1 microglial inhibitory receptor as a therapeutic target in the MPTP model of Parkinson's disease

Abstract

Background: It is suggested that neuroinflammation, in which activated microglial cells play a relevant role, contributes to the development of Parkinson's disease (PD). Consequently, the modulation of microglial activation is a potential therapeutic target to be taken into account to act against the dopaminergic neurodegeneration occurring in this neurological disorder. Several soluble and membrane-associated inhibitory mechanisms contribute to maintaining microglial cells in a quiescent/surveillant phenotype in physiological conditions. However, the presence of activated microglial cells in the brain in PD patients suggests that these mechanisms have been somehow overloaded. We focused our interest on one of the membrane-associated mechanisms, the CD200-CD200R1 ligand-receptor pair.

Methods: The acute MPTP experimental mouse model of PD was used to study the temporal pattern of mRNA expression of CD200 and CD200R1 in the context of MPTP-induced dopaminergic neurodegeneration and neuroinflammation. Dopaminergic damage was assessed by tyrosine hydroxylase (TH) immunoreactivity, and neuroinflammation was evaluated by the mRNA expression of inflammatory markers and IBA1 and GFAP immunohistochemistry. The effect of the modulation of the CD200-CD200R1 system on MPTP-induced damage was determined by using a CD200R1 agonist or CD200 KO mice.

Results: MPTP administration resulted in a progressive decrease in TH-positive fibres in the striatum and TH-positive neurons in the substantia nigra pars compacta, which were accompanied by transient astrogliosis, microgliosis and expression of pro- and anti-inflammatory markers. CD200 mRNA levels rapidly decreased in the ventral midbrain after MPTP treatment, while a transient decrease of CD200R1 mRNA expression was repeatedly observed in this brain area at earlier and later phases. By contrast, a transient increase in CD200R1 expression was observed in striatum. The administration of a CD200R1 agonist resulted in the inhibition of MPTP-induced dopaminergic neurodegeneration, while microglial cells showed signs of earlier activation in CD200-deficient mice.

Conclusions: Collectively, these findings provide evidence for a correlation between CD200-CD200R1 alterations, glial activation and neuronal loss. CD200R1 stimulation reduces MPTP-induced loss of dopaminergic neurons, and CD200 deficiency results in earlier microglial activation, suggesting that the potentiation of CD200R1 signalling is a possible approach to controlling neuroinflammation and neuronal death in PD.

Keywords: CD200 KO mice; CD200-CD200R1 system; CD200Fc; Glia; Immune response; MPTP; Microglia; Neuroinflammation; Parkinson’s disease.

Fig. 1

Time course of dopaminergic degeneration in the acute MPTP mouse model. a Experimental design. Mice were injected intraperitoneally with saline or MPTP (20 mg/kg) every 2 h to a total of 4 doses in 1 day. Animals were sacrificed at 2 h and at 1, 2, 4, and 7 days (d) after the last injection. Right hemispheres were fixed for immunohistochemistry (IHC) and left hemispheres were processed for gene expression analysis (RNA). b TH immunostaining in striatum and substantia nigra pars compacta (SNpc). c Optical densitometry of striatal TH-positive dopaminergic fibers. d Stereological cell counts of dopaminergic neurons in SNpc. Bars are means + SEM of seven to eight mice per group. **p < 0.01 and ***p < 0.001 vs. saline; one-way ANOVA and Newman-Keuls post hoc test. Scale bars: 500 μm (striatum) and 200 μm (SNpc)

Fig. 2

Glial activation associated with MPTP-induced dopaminergic degeneration. a Representative photomicrographs of IBA1- and GFAP-immunostained striatum of control mice administered with saline and MPTP-injected mice at the indicated time points after MPTP injections. b Representative photomicrographs of IBA1- and GFAP-immunostained substantia nigra pars compacta (SNpc) of control mice administered with saline and MPTP-injected mice at the indicated time points after MPTP injection. Scale bars: 100 μm (striatum), 200 um (SNpc) and 50 μm (insets)

Fig. 3

Effect of MPTP administration on the temporal pattern of mRNA expression of molecules involved in the brain inflammatory response. Mice were administered with saline (S) or MPTP and sacrificed at 2 h, and at 1, 2, 4 and 7 days (d) after the last MPTP injection. mRNA expression of the pro-inflammatory cytokines IL1β, IL6 and TNFα (a, b), the pro-inflammatory enzymes iNOS, COX2 and gp91phox (c, d), the anti-inflammatory cytokines IL10 and TGFβ (e, f), the anti-inflammatory markers Arg1, MR and Nrf2 (g, h) and the components of the CD200-CD200R1 inhibitory system (the ligands CD200full and CD200tr and the receptor CD200R1) (i, j) in striatum (St, left column) and ventral midbrain (MV, right column) of saline- and MPTP-treated mice. Gapdh and βactin were used as reference genes. Bars are means + SEM of seven to eight mice per experimental group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. saline control; one-way ANOVA and Newman-Keuls post hoc test

Fig. 4

Effect of CD200R1 agonist administration on dopaminergic degeneration in the acute MPTP mouse model. a Experimental design. Mice were injected intraperitoneally with saline or MPTP (20 mg/kg) every 2 h to a total of 4 doses in 1 day. In mice treated with the CD200R1 agonist, 1.8 mg/kg or 3.6 mg/kg CD200Fc were intraperitoneally administered twice, 30 min before the first MPTP injection and 24 h after the last one. Isotype administration regimen was the same as for CD200Fc. Mice were sacrificed 7 days (d) following the last MPTP injection and brains were fixed for immunohistochemistry (IHC). b TH immunostaining in striatum and optical densitometry of striatal TH-positive dopaminergic fibers of saline, MPTP, CD200Fc + MPTP and isotype+MPTP, considering the lowest dose (1.8 mg/kg) (c) and the highest dose (3.6 mg/kg) (d) of CD200Fc. e TH immunostaining and stereological cell counts of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of saline, MPTP, CD200Fc + MPTP and isotype+MPTP, considering the lowest dose (1.8 mg/kg) (f) and the highest dose (3.6 mg/kg) (g) of CD200Fc. Bars are means + SEM of five to eight mice per experimental group. **p < 0.01 and ***p < 0.001 vs. saline; one-way ANOVA and Newman-Keuls post hoc test. Scale bars: 500 μm (striatum) and 200 μm (SNpc)

Fig. 5

Effect of CD200 deficiency on MPTP-induced dopaminergic neurodegeneration and microglial activation at day 7 after administration. Mice were injected intraperitoneally with saline or MPTP (18 mg/kg) every 2 h to a total of 4 doses in 1 day. Mice were sacrificed 7 days following the last MPTP injection and brains were fixed for immunohistochemistry. a TH immunostaining in striatum and optical densitometry of striatal TH-positive dopaminergic fibers of saline- and MPTP-treated CD200 +/+ and CD200 −/− mice. b TH immunostaining and stereological cell counts of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of saline- and MPTP-treated CD200 +/+ and CD200 −/− mice. c Representative photomicrographs of IBA1-immunostained substantia nigra (SN) of CD200 +/+ and CD200 −/− mice treated with saline or MPTP. Quantification of the IBA1-labelled area (d) and the immunolabelling intensity of IBA1-positive cells (e) in total SN and SNpc of saline- and MPTP-treated CD200 +/+ and CD200 −/− mice. Bars are means + SEM of five to eight mice per experimental group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. saline; two-way ANOVA and Bonferroni post hoc test. Scale bars: 500 μm (striatum) and 200 μm (SN and SNpc)

Fig. 6

Effect of CD200 deficiency on MPTP-induced dopaminergic neurodegeneration and microglial activation at day 1 after administration. Mice were injected intraperitoneally with saline or MPTP (18 mg/kg) every 2 h to a total of 4 doses in one day. Mice were sacrificed 1 day after the last MPTP injection and brains were fixed for immunohistochemistry. a TH immunostaining in striatum and optical densitometry of striatal TH-positive dopaminergic fibers of saline- and MPTP-treated CD200 +/+ and CD200 −/− mice. b TH immunostaining and stereological cell counts of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of saline- and MPTP-treated CD200 +/+ and CD200 −/− mice. c Representative photomicrographs of IBA1-immunostained substantia nigra (SN) of CD200 +/+ and CD200 −/− mice treated with saline or MPTP. Quantification of the IBA1-labelled area (d), the immunolabelling intensity of IBA1-positive cells (e) and the total number of IBA1-positive cells in total SN and SNpc of saline- and MPTP-treated CD200 +/+ and CD200 −/− mice. Bars are means + SEM of five to eleven mice per experimental group. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. saline; ##p < 0.01 vs. CD200 +/+ MPTP-treated mice; two-way ANOVA and Bonferroni post hoc test. Scale bars: 500 μm (striatum) and 200 μm (SN and SNpc)