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, 39 (19), 3752-3769

Translocator Protein Ligand Protects Against Neurodegeneration in the MPTP Mouse Model of Parkinsonism

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Translocator Protein Ligand Protects Against Neurodegeneration in the MPTP Mouse Model of Parkinsonism

Jing Gong et al. J Neurosci.

Abstract

Parkinson's disease is the second most common neurodegenerative disease, after Alzheimer's disease. Parkinson's disease is a movement disorder with characteristic motor features that arise due to the loss of dopaminergic neurons from the substantia nigra. Although symptomatic treatment by the dopamine precursor levodopa and dopamine agonists can improve motor symptoms, no disease-modifying therapy exists yet. Here, we show that Emapunil (AC-5216, XBD-173), a synthetic ligand of the translocator protein 18, ameliorates degeneration of dopaminergic neurons, preserves striatal dopamine metabolism, and prevents motor dysfunction in female mice treated with the MPTP, as a model of parkinsonism. We found that Emapunil modulates the inositol requiring kinase 1α (IRE α)/X-box binding protein 1 (XBP1) unfolded protein response pathway and induces a shift from pro-inflammatory toward anti-inflammatory microglia activation. Previously, Emapunil was shown to cross the blood-brain barrier and to be safe and well tolerated in a Phase II clinical trial. Therefore, our data suggest that Emapunil may be a promising approach in the treatment of Parkinson's disease.SIGNIFICANCE STATEMENT Our study reveals a beneficial effect of Emapunil on dopaminergic neuron survival, dopamine metabolism, and motor phenotype in the MPTP mouse model of parkinsonism. In addition, our work uncovers molecular networks which mediate neuroprotective effects of Emapunil, including microglial activation state and unfolded protein response pathways. These findings not only contribute to our understanding of biological mechanisms of translocator protein 18 (TSPO) function but also indicate that translocator protein 18 may be a promising therapeutic target. We thus propose to further validate Emapunil in other Parkinson's disease mouse models and subsequently in clinical trials to treat Parkinson's disease.

Keywords: MPTP; Parkinson's disease; TSPO; microglia; neurodegeneration.

Figures

Figure 1.
Figure 1.
Reagents and conditions. a, NaOEt, EtOH, 0°C-80°C, 4 h, 81%. b, POCl3, 90°C, 4 h, 99%. c, Glycine, Et3N, EtOH, 78°C, 4 h, 100%. d, N-Ethylbenzylamine, PyBOP, Et3N, DMF, 22°C, 2 h, 100%. e, NaOH, EtOH, water, 78°C, 2 h, 82%. f, Diphenyl phosphorazidate, Et3N, DMF, 100°C, 6 h, 55%. g, MeI, NaH, DMF, 22°C, 3 h, 78%.
Figure 2.
Figure 2.
Experimental design. Eight-week-old female C57BL/6JRj mice were assigned to three different experimental treatment groups: Top, NaCl group. Middle, MPTP + DMSO group. Bottom, MPTP + Emapunil group. Animals either received NaCl or 30 mg/kg body weight of MPTP by intraperitoneal injection once daily on 5 subsequent days (days 1–5). Animals in the MPTP + Emapunil group were additionally treated with intraperitoneal injections of 50 mg/kg body weight of Emapunil every second day, starting from day 1 until day 15. Five animals of each group were killed on day 3; striatum as well as substantia nigra were prepared and shock-frozen for further analysis by qPCR, HPLC, and transcriptome. The remaining animals underwent motor function tests on days 11–13 (pole test) and on day 14 (cylinder test). Animals were perfused on day 15, and brains were cryo-frozen for further stereological analysis.
Figure 3.
Figure 3.
Emapunil ameliorates MPTP-induced dopaminergic neuron loss. A, Representative images of the substantia nigra of mice treated with NaCl (top), MPTP (middle), or MPTP and Emapunil (bottom) (anti-TH antibody staining). Scale bar, 200 μm. B, Dopaminergic neurons were quantified as the total number of TH-immunoreactive neurons. C, The number of Nissl-stained neurons was quantified in the NaCl, MPTP, or MPTP and Emapunil treatment groups. One-way ANOVA, in between-group difference: p < 0.0001 (both in TH and Nissl stained neurons). *p < 0.05; ***p < 0.001; Tukey's post hoc test.
Figure 4.
Figure 4.
Emapunil treatment restores motor function. A, Cylinder test of mice treated with NaCl (black bars), MPTP (red bars), or MPTP + Emapunil (green bars) 14 d after first MPTP application. Histograph represents the percentage of rears against the cylinder wall, either as free rears without paw support or rears supported with only the left, only the right or with both paws. No significant between-group difference was observed for right or left paw only assisted rears. One-way ANOVA, in between-group difference: p = 0.000108 (free rears) and p = 0.003677 (support with both paws). *p < 0.05; **p < 0.01; ***p < 0.001; Tukey's post hoc test. B, Pole test performance 11–13 d after the initial MPTP dose. Histograph represents the time taken to orient downward at the top of the pole. One-way ANOVA, in between-group difference: p = 0.04. *p < 0.05 (Tukey's post hoc test).
Figure 5.
Figure 5.
HPLC analysis of dopamine and its metabolites in the striatum. A, Dopamine. B, DOPAC. C, Metabolic ratio (HVA + DOPAC)/dopamine. One-way ANOVA, in between-group difference: p < 0.0001 (dopamine), p = 0.02 (DOPAC), and p = 0.0008 (metabolic ratio). *p < 0.05; **p < 0.01; ***p < 0.001; Tukey's post hoc test.
Figure 6.
Figure 6.
Emapunil mitigates ER stress. A, qPCR analysis of relative mRNA expression levels of unspliced Xbp1. B, qPCR analysis of relative mRNA expression levels of spliced Xbp1s. C, Ratio of spliced Xbp1s/unspliced Xbp1 normalized to NaCl control. One-way ANOVA, in between-group difference: p = 0.25329 (Xbp1), p < 0.0001 (spliced Xbp1s), and p = 0.003151 (ratio Xbp1s/Xbp1). **p < 0.01; ***p < 0.001; Tukey's post hoc test. D, Cell toxicity was measured by adenylate kinase release (relative ToxiLight luminescence) in differentiated LUHMES cells treated with DMSO, MPP+, or rotenone and with or without addition of Emapunil. Two-way ANOVA, in all interaction comparisons: p < 0.0001. *p < 0.05; ***p < 0.001; Holm–Sidak post hoc test. E, XBP1s/XBP1 ratios in LUHMES cells. One-way ANOVA, in between-group difference: p < 0.0001. **p < 0.01; ***p < 0.001; Tukey's post hoc test.
Figure 7.
Figure 7.
TSPO expression in dopaminergic neurons. A, Immunofluorescence staining of 8-week-old WT control mice (substantia nigra). Sections were stained with antibodies against TSPO (left, green) and TH (middle, red). Right, Merged image. Scale bar, 20 μm. B, Immunofluorescence staining of LUHMES cells. Left, DAPI (blue), antibody against TSPO (green), antibody against β-III-tubulin (red). Right, Merged image. Scale bar, 50 μm. C, Western blot analysis of TSPO expression in LUHMES cells.
Figure 8.
Figure 8.
TSPO siRNA treatment decreases TSPO expression levels. A, Western blot analysis of TSPO, β-actin, and TH levels in LUHMES cells, with and without siRNA directed against TSPO. B, Quantification of TSPO levels (Western blot analysis from A) normalized to β-actin (loading control). Two-tailed unpaired t test (Welch's t test): p = 0.0048. **p < 0.005. C, Quantification of TH levels (Western blot analysis from A) normalized to β-actin (loading control). Two-tailed unpaired t test (Welch's t test): p = 0.412. n.s. = not significant.
Figure 9.
Figure 9.
The effects of Emapunil depend on TSPO expression. A, Cytotoxicity measured by adenylate kinase release (ToxiLight luminescence) in differentiated LUHMES cells treated with either TSPO or control siRNA. Experimental groups: DMSO, 10 μm rotenone, 5 μm Emapunil, 10 μm rotenone plus 5 μm Emapunil. Two-way ANOVA: F(3,54) = 3.365, p = 0.0251. **p < 0.005; ***p < 0.001; Tukey post hoc test. B, Cytotoxicity (ToxiLight luminescence assay) in differentiated LUHMES cells with increasing rotenone concentrations. One-way ANOVA, in between-group difference: p < 0.0001. ***p < 0.001 (Tukey post hoc test). C, qPCR analysis of spliced XBP1s/unspliced XBP1 in differentiated LUHMES cells treated with TSPO or control siRNA. Experimental groups as in A. Two-way ANOVA: F(3,82) = 6.458, p = 0.0006. **p < 0.005; ***p < 0.001; Tukey post hoc test. n.s. = not significant.
Figure 10.
Figure 10.
Emapunil prevents microgliosis and astrogliosis in MPTP animals. A, IBA1 (green) immunohistochemistry of striatal sections from NaCl, MPTP + DMSO, or MPTP + Emapunil-treated mice, killed at day 15. B, GFAP (red) immunohistochemistry of striatal sections from NaCl, MPTP + DMSO, or MPTP + Emapunil-treated mice, killed at day 15. C, Quantification of IBA1-positive cells (striatum). D, GFAP-positive cells (striatum). Data are total numbers/mm2; three sections from each animal were analyzed with n = 6 or 7 animals per group. One-way ANOVA, in between-group differences: p < 0.0001 (both in IBA1 and GFAP quantification). ***p < 0.001 (Tukey's post hoc test). Scale bars: A, 50 μm; B, 100 μm. n.s. = not significant.
Figure 11.
Figure 11.
Emapunil mediates a shift from pro-inflammatory to anti-inflammatory gene expression. Striatal RNA was isolated on day 3 from mice treated with NaCl (black bars), MPTP + DMSO (red bars), or MPTP + Emapunil (green bars) (n = 5 per experimental group). qPCR analysis of mRNA expression: A, Iba1. B, Tspo. C, Pro-inflammatory cytokines and chemokines. D, Anti-inflammatory cytokines and chemokines. Histographs represent relative mRNA levels normalized to NaCl control condition. One-way ANOVA, in between-group difference: p = 0.000163 (Iba1), p = 0.001157 (Tspo), p = 0.000151 (Tnfa), p < 0.0001 (Cxcl10), p = 0.004103 (Il1b), p = 0.81803 (Mpa2l), p = 0.0039 (Il6), p = 0.019653 (Nos2), p = 0.000377 (Cox2), p = 0.015957 (Arg1), p = 0.008185 (Il10), p = 0.0895 (Il4), p = 0.00057 (Mrc1), p = 0.000336 (Ym1), p = 0.000169 (Fizz2). *p < 0.05; **p < 0.01; ***p < 0.001; Tukey's post hoc test. n.s. = not significant.
Figure 12.
Figure 12.
Effects of MPTP and Emapunil on the transcriptome. A, Venn diagram showing the overlap between the transcriptional response to MPTP and Emapunil as well as genes that are only affected by MPTP treatment or only affected by additional Emapunil treatment (MPTP + Emapunil). B, Individual traces for the 183 genes that are significant in the MPTP group alone (i.e., attenuated by Emapunil treatment). Right, The most salient gene ontology (GO) categories for upregulated and downregulated genes within those 183 are represented. C, Network of GO terms associated with Emapunil-specific genes (i.e., those 1630 in the Venn diagram from A that are only changed in the MPTP + Emapunil treatment group but not in the MPTP group). LFC, Log fold change.

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