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Comparative Study
. 2007 Mar 21;27(12):3328-37.
doi: 10.1523/JNEUROSCI.5321-06.2007.

Involvement of interferon-gamma in microglial-mediated loss of dopaminergic neurons

Matthew P Mount  1 , Arman Lira, David Grimes, Patrice D Smith, Sylvie Faucher, Ruth Slack, Hymie Anisman, Shawn Hayley, David S Park
Affiliations
Comparative Study

Involvement of interferon-gamma in microglial-mediated loss of dopaminergic neurons

Matthew P Mount et al. J Neurosci. .

Abstract

Growing evidence implicates microglia in the loss of dopaminergic neurons in Parkinson's disease (PD). However, factors mediating microglial activation in PD are poorly understood. Proinflammatory cytokines, such as interferon-gamma (IFN-gamma), orchestrate the actions of microglia. We report here that PD patients express significantly elevated levels of IFN-gamma in their blood plasma. After this initial finding, we found that IFN-gamma-deficient mice displayed attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced substantia nigra pars compacta dopaminergic cell loss along with reduced loss of striatal tyrosine hydroxylase and dopamine transporter fiber density. MPTP-induced depletion of striatal dopamine and its metabolite DOPAC (3,4-dihydroxyphenylacetic acid), as well as deltaFosB, a marker of postsynaptic dysfunction, were also attenuated in these knock-out mice. Consistent with the role for IFN-gamma in microglial activation, MPTP-induced morphological activation of microglia was abrogated compared with wild-type mice. To examine more mechanistically the role of IFN-gamma in microglial activation, we evaluated the interactions between microglia and dopaminergic neurons in an in vitro mixed microglia/midbrain neuron rotenone-induced death paradigm. In this in vitro paradigm, dopaminergic neurons are selectively damaged by rotenone. Exogenous IFN-gamma ligand alone and without rotenone resulted in dopaminergic cell loss, but only in the presence of microglia. The addition of an IFN-gamma neutralizing antibody attenuated neuronal loss as a result of rotenone treatment. The presence of only wild-type microglia and not those deficient in IFN-gamma receptor elicited significant dopaminergic cell loss when exposed to rotenone. Neurons deficient in IFN-gamma receptor, however, did not display increased resistance to death. Finally, levels of IFN-gamma message increased in microglia in response to rotenone. Together, these data suggest that IFN-gamma participates in death of dopaminergic neurons by regulating microglial activity.

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Figures

Figure 1.
Figure 1.
PD patients display elevated IFN-γ plasma levels relative to non-PD patients. Blood plasma from 13 PD and 7 control patients was analyzed by a multiplex bead assay system for several cytokines. Results for IFN-γ concentrations are provided here in dot plot representation (two-tailed t test, **p < 0.01).
Figure 2.
Figure 2.
IFN-γ-deficient mice display protection of dopaminergic cell bodies and striatal terminals of MPTP-induced degeneration of SNc dopaminergic neurons. A, Representative photomicrographs illustrating TH immunoreactivity in the ventral midbrain SNc of the indicated non-littermate treatment groups, as indicated in Materials and Methods. B, Quantification of TH+ neurons using Abercrombie correction, as described in Materials and Methods. C, Quantification of cresyl violet-stained cells of the SNc (MTN level). D, Representative photomicrographs of striatal sections stained with TH from the animal groups as described in A. Magnified images are included in the insets of D–J. E, Quantification of optical density of TH striatal fibers. F, Representative photomicrographs illustrating DAT immunoreactivity in the striatum of indicated treatment groups as in A. G, Quantification of optical density of striatal DAT-stained fiber density. H, Representative photomicrographs illustrating TH immunoreactivity in the SNc of the indicated littermate treatment animal groups. Staining is more intense here than in A because of increased section thickness required for stereological analyses. I, Quantification of TH+ neurons, in littermate animals by stereology, as described in Materials and Methods. J, Representative photomicrographs of striatal TH fiber density of indicated littermate treatment groups. K, Quantification of OD of striatal TH-stained fiber density of indicated littermates. Error bars represent mean ± SEM. ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001; n = 6–8 animals per group. OD, Optical density; Sal, saline.
Figure 3.
Figure 3.
IFN-γ KO mice display reduced striatal ΔFosB, a marker for postsynaptic changes in the denervated striatum, as well as attenuated DA and DOPAC. A, Representative photomicrographs showing ΔFosB staining in the striatum of mice treated as indicated. Sal, Saline. B, Quantification of ΔFosB-positive cells/nuclei. Str, striatum. C, D, Levels of DA (C) and its metabolite DOPAC (D) in the striatum were analyzed using HPLC on 14-d-old tissue after saline/MPTP injection. Error bars represent mean ± SEM. ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001; n = 6 animals per group.
Figure 4.
Figure 4.
Analysis of active microglia using anti-CD11b. A, Representative photomicrographs illustrating CD11b immunoreactivity in the SNc (MTN) of WT and KO animals treated with either saline (Sal) or MPTP. Arrows indicate resting CD11b-positive microglia (WT/Sal) and activated microglia (WT/MPTP). B, Quantification of morphologically active microglia. Magnified images of each are provided in the insets of A (WT/Sal and WT/MPTP). Error bars represent mean ± SEM (ANOVA, **p < 0.01; n = 6 animals per group).
Figure 5.
Figure 5.
IFN-γ requirement in a coculture model of dopaminergic cell loss induced by rotenone in vitro. A, Neutralizing IFN-γ antibody protects dopaminergic neurons from rotenone-induced death in the presence of microglia. Seven-day-old WT neuron-enriched midbrain cultures were cocultured with and without WT microglia in the presence or absence of rotenone and with control or IFN-γ neutralizing antibody, as described in Materials and Methods and as shown. The number of live TH+ neurons was assessed as described in Materials and Methods. *p < 0.05, ***p < 0.001 relative to coculture with control IgG (two-tailed t test). B, Seven-day-old neuron-enriched midbrain cultures supplemented with or without microglia were subjected to IFN-γ ligand (nanograms per milliliter) applications. The cultures were fixed 7 d later and stained with TH and Hoechst. ***p < 0.001 compared with control, **p < 0.01 (two-tailed t test; n = 3). C, Dopaminergic neurons treated with rotenone are resistant to death in the presence of microglia lacking IFN-γ receptor. Dopaminergic neurons used were similar to those in A, except WT neurons were cultured in the presence of either WT or IFN-γ receptor (IFN- R)-deficient microglia. ‡p < 0.05 relative to IFN-γ receptor KO (two-tailed t test; n = 3). D, Representative immunofluorescence of cocultures quantified in C, stained with TH antibody are as follows: a, WT mesencephalic neurons; b, WT mesencephalic neurons treated with rotenone; c, WT mesencephalic neurons and microglia treated with rotenone; d, WT mesencephalic neurons and IFN-γ receptor KO microglia treated with rotenone. E, WT microglia-enriched cultures were treated with rotenone for 0, 1, and 3 d. One week after plating, microglia were collected, RNA was extracted, and RT-PCR was performed for IFN-γ and S12 control. Densitometric analysis was performed with values normalized to each respective S12 signal and further normalized to 0 d value. +, IFN-γ-positive RNA control sample obtained from CD4+ T-cells. Error bars represent mean ± SEM.
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