IFN-γ signaling, with the synergistic contribution of TNF-α, mediates cell specific microglial and astroglial activation in experimental models of Parkinson's disease

Abstract

To through light on the mechanisms underlying the stimulation and persistence of glial cell activation in Parkinsonism, we investigate the function of IFN-γ and TNF-α in experimental models of Parkinson's disease and analyze their relation with local glial cell activation. It was found that IFN-γ and TNF-α remained higher over the years in the serum and CNS of chronic Parkinsonian macaques than in untreated animals, accompanied by sustained glial activation (microglia and astroglia) in the substantia nigra pars compacta. Importantly, Parkinsonian monkeys showed persistent and increasing levels of IFN-γR signaling in both microglial and astroglial cells. In addition, experiments performed in IFN-γ and TNF-α KO mice treated with MPTP revealed that, even before dopaminergic cell death can be observed, the presence of IFN-γ and TNF-α is crucial for microglial and astroglial activation, and, together, they have an important synergistic role. Both cytokines were necessary for the full level of activation to be attained in both microglial and astroglial cells. These results demonstrate that IFN-γ signaling, together with the contribution of TNF-α, have a critical and cell-specific role in stimulating and maintaining glial cell activation in Parkinsonism.

Figure 1

Persistent increase of IFN-γ and TNF-α in chronic Parkinsonian monkeys. (a–d) Artist's sketch (a) of the typical gait posture of a normal monkey (control) compared with the characteristic Parkinsonian posture (Parkinsonian). Parkinsonian monkey posture is characterized by curvature of the trunk and rigidity of the limbs, together with rigidity of the tail (drawings by C.B). (b) Graphs of the motor score impairment reached by the MPTP-treated monkeys included in the study. Some animals, despite MPTP administration, did not show symptoms (asymptomatic). (c) Levels of IFN-γ measured by ELISA in serum increase in Parkinsonian monkeys. (d) Levels of TNF-α measured by ELISA in serum are increased in Parkinsonian monkeys. ^*P<0.05 respect to non-Parkinsonian

Figure 2

Increase of IFN-γ in the SNpc of Parkinsonian monkeys. (a–f): Increase of IFN-γ⁺ cells in brain sections of the SNpc of Parkinsonian monkeys. (a) Confocal images of immunofluorescence against IFN-γ revealed a higher number of IFN-γ⁺ cells in MPTP-treated animals. Scale bar: 100 μm. (b) The density of cells was estimated by stereological criteria and a significant increase in IFN-γ⁺ cells was observed in MPTP-treated animals. (c) Western blot of IFN-γ from monkey brain samples. Tissue of the SNpc from seven different monkeys (1–7) is shown. The Parkinsonian motor score of the monkeys is indicated between brackets. GAPDH was used as house-keeping protein. The analysis of the optical density of all the studied monkeys, normalized with GAPDH, is represented in Table 2. (d) Multiple fluorescence labeling revealed that IFN-γ (red) co-localize with microglial marker Iba-1 (green) (1). However, astrocyte marker GFAP (green) did not co-localize with IFN-γ-expressing cells (red) (2). DAPI staining was used to mark the cell nuclei (Blue). Scale bar: 50 μm. (e) Higher magnification of two representative IFN-γ⁺ microglial cells. Some of the microglial cells with resting phenotype (1) presented IFN-γ staining mainly confined to the cytoplasm (red) surrounding the cell nucleus (Blue). Other microglial cells with activated phenotype (2) presented IFN-γ staining through the whole cytoplasm. (f) Percentage of IFN-γ-expressing microglia (black pie portion (Iba-1⁺/IFN-γ⁺ cells)) over the rest of the microglial population (gray pie portion (Iba-1⁺/IFN-γ⁻ cells)) in the three group of animals. ^*P<0.05 (one-way ANOVA and Tukey's test)

Figure 3

Microglial and astroglial cells express IFN-γR in the SNpc of MPTP-treated monkeys. (a and b) Sections of the SNpc of MPTP-treated monkeys were immuno-stained for IFN-γR (Green), combined with either astrocyte marker GFAP (Red in a) or microglial marker Iba-1 (Red in b) and counterstained with DAPI (Blue). Scale bars: in a, 50 μm; in b, 40 μm

Figure 4

Persistent phosphorylation of STAT1 (pSTAT1) in the SNpc of chronic Parkinsonian monkeys. (a–e) Panel (a) shows confocal images of pSTAT1 in a representative image of the SNpc of a control monkey, asymptomatic and Parkinsonian monkey. DAPI staining is combined to observe the cell nuclei (Blue). High levels of pSTAT1 staining (Red) can be seen in Parkinsonian monkeys. Scale bar: 100 μm. (b) Quantification of pSTAT1⁺ cells in the SNpc of control, asymptomatic and Parkinsonian monkeys. A strongly significant increase can be observed in Parkinsonian monkeys. ^**P<0.01 (one-way ANOVA and Tukey's test). (c) The pSTAT1 immunostaining in the SNpc identified astroglia-like cells (1) and microglia-like cells (2). Scale bar: 30 μm. (d) The pSTAT1 immunostaining (Red) was combined with GFAP or Iba-1 antibodies (Green) and both astrocytes (GFAP) and microglia (Iba-1) were seen to co-localize with pSTAT1. Nuclei were stained with DAPI (Blue). Scale bar: 20 μm. (e) Illustration of the IFN-γ signaling, characterized by the phosphorylation of STAT1, through the activation of IFN-γ receptor (IFN-γR) in a microglial cell and an astrocyte

Figure 5

Persistent increase of TNF-α in the CNS of chronic Parkinsonian monkeys. (a–f) Increase of TNF-α⁺ cells in the SNpc of Parkinsonian monkeys. (a) Confocal images of immunofluorescence against TNF-α revealed higher levels in Parkinsonian animals. Scale bar: 100 μm. (b) The density of cells was estimated by stereological criteria and a significant increase of TNF-α⁺ cells were observed in Parkinsonian animals. (c) Western blot of TNF-α from monkey brain samples. Tissue of the SNpc from seven different monkeys (1–7) is shown. The Parkinsonian motor score of the monkeys is indicated between brackets. GAPDH was used as house-keeping protein. TNF-α was specifically detected in the expected band in the SNpc of MPTP-treated monkeys. The quantification of the optical density of all the analyzed monkeys, normalized with GAPDH, is represented in Table 2. (d) Multiple fluorescence labeling revealed that TNF-α (red) did not co-localize with microglial marker Iba-1 (green) (1). However, astrocyte marker GFAP (green) co-localized with TNF-α-expressing cells (red) (2). DAPI staining was used to mark the cell nuclei (Blue). Scale bar: 25 μm. (e) Higher magnification of a representative TNF-α⁺ astrocyte. (f) Percentage of TNF-α-expressing astrocytes (black pie portion (GFAP⁺/TNF-α⁺ cells)) over the rest of the astroglial population (gray pie portion (GFAP⁺/TNF-α⁻ cells)) in the three group of animals. ^*P<0.05 (one-way ANOVA and Tukey's test)

Figure 6

IFN-γ and TNF-α have a cell specific function in microglial and astroglial activation. (a–f) Activation of the microglia depends on IFN-γ and activation of the astrocytes depends on TNF-α. (a) Quantification of TH⁺ neurons in the SNpc revealed no significant reduction of dopaminergic neurons in any of the experimental groups 24 h after MPTP. (b) Quantification of the number of F4/80⁺ microglial cells and GFAP⁺ astroglial cells. In the absence of IFN-γ, no increase in the number of F4/80⁺ or GFAP⁺ cells was observed after MPTP treatment. In the absence of TNF-α, a slight increase of F4/80⁺ cells was observed, but no changes were noted in the number of GFAP⁺ cells. ^*P<0.05 (one-way ANOVA and Tukey's test). (c) Confocal pictures of microglial cells (Iba-1) and astroglial cells (GFAP) in the SNpc of wild type mice (C57 BL6), IFN-γ KO mice (IFNγ(−/−)) and TNF-α KO mice (TNF-α(−/−)). Scale bar: 30 μm. (d) Quantification of the area occupied by Iba-1 demonstrates that the size of microglial cells (Iba-1) increases significantly after MPTP treatment. However, in the absence of IFN-γ, the microglial cells do not change their size. In TNF-α(−/−) mice, microglial cells also increase their size, but to a lesser extent. Quantification of the area occupied by GFAP demonstrated that the size of astroglial cells increases after MPTP treatment. In the absence of IFN-γ, astroglial cells also increase their size after MPTP; however, in the absence of TNF-α, astroglial cells remain unchanged after MPTP. ^*P<0.01 with respect to control saline values, ^†P<0.01 with respect to wild type C57 MPTP (one-way ANOVA and Tukey's test). (e) Quantification of primary branches (1ary), secondary branches (2ary) and terminal tips (T) in microglial (Iba-1) and astroglial cells (GFAP). Both microglial and astroglial cells change their phenotype 24 h after MPTP treatment in wild-type animals (C57BL6). In animals without IFN-γ (IFNγ(−/−)), microglial cells show no changes in primary branches, but increased numbers of secondary branches and terminal tips. In TNF-α KO animals (TNFα(−/−)), microglial cells show increased numbers of primary, secondary branches and T. However, astroglial cells remain unchanged in TNF-α(−/−) mice 24 h after MPTP. ^*P<0.01, ^▾P<0.05 (one-way ANOVA and Dunnett's test)