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. 2014 Nov 17;143(2):360–373. doi: 10.1093/toxsci/kfu236

Novel Para-Phenyl Substituted Diindolylmethanes Protect Against MPTP Neurotoxicity and Suppress Glial Activation in a Mouse Model of Parkinson’s Disease

Briana R De Miranda *, Katriana A Popichak *, Sean L Hammond *, James A Miller *, Stephen Safe †,‡, Ronald B Tjalkens *,1
PMCID: PMC4306720  PMID: 25406165

Abstract

The orphan nuclear receptor NR4A2 (Nurr1) constitutively regulates inflammatory gene expression in glial cells by suppressing DNA binding activity of NF-κB. We recently reported that novel 1,1-bis(3′-indolyl)-1-(p-substitutedphenyl)methane (C-DIM) compounds that activate NR4A family nuclear receptors in cancer lines also suppress inflammatory gene expression in primary astrocytes and prevent loss of dopaminergic neurons in mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp). In this study, we postulated that the basis for this neuroprotection involves blockade of glial activation and subsequent expression of NF-κB-regulated inflammatory genes. To examine this mechanism, we treated transgenic NF-κB/EGFP reporter mice with MPTPp for 7 days (MPTPp7d) followed by daily oral gavage with either vehicle (corn oil; MPTPp14d) or C-DIMs containing p-methoxyphenyl (C-DIM5), p-hydroxyphenyl (C-DIM8), or p-chlorophenyl (C-DIM12) groups. Each compound conferred significant protection against progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), even when given after 7 days of dosing with MPTPp. C-DIM12 had the greatest neuroprotective activity in MPTPp-treated mice, and was also the most potent compound in suppressing activation of microglia and astrocytes, expression of cytokines and chemokines in quantitative polymerase chain reaction (qPCR) array studies, and in reducing expression of NF-κB/EGFP in the SN. C-DIM12 prevented nuclear export of Nurr1 in dopaminergic neurons and enhanced expression of the Nurr1-regulated proteins tyrosine hydroxylase and the dopamine transporter. These data indicate that NR4A-active C-DIM compounds protect against loss of dopamine neurons in the MPTPp model of PD by preventing glial-mediated neuronal injury and by supporting a dopaminergic phenotype in TH-positive neurons in the SNpc.

Keywords: Parkinson’s disease, neuroinflammation, microglia, orphan nuclear receptors

Glial-derived inflammatory mediators exacerbates loss of dopaminergic neurons in PD and contributes to the deterioration of neurological function that occurs as the disease progresses (Glass et al., 2010; Hirsch and Hunot, 2009; Nolan et al., 2013; Tansey and Goldberg, 2010). Although it remains unclear whether neuroinflammation is an early etiologic factor in PD evidence indates that microglia and astrocytes are directly involved in inflammatory injury to neurons (Hirsch and Hunot, 2009; Tansey and Goldberg, 2010). Lack of available drugs to prevent neuroinflammatory injury is a major obstacle to PD therapy.

NF-κB is a major transcriptional activator of inflammatory genes in glia and is constitutively suppressed under resting conditions by both cytoplasmic proteins, such as IκBα, the inhibitory subunit of NF-κB, and by nuclear co-repressors, such as NCoR2, CoREST, and HDAC2/3 (Carbone et al., 2008; Ghisletti et al., 2009). Overproduction of NF-κB-regulated inflammatory mediators such as nitric oxide (NO), cytokines (TNFα, IL-1β), and chemokines (CCL2, CCL5) is acutely damaging to neurons (Bianchi et al., 2010; Thompson and Van Eldik, 2009). These signaling pathways are therefore tightly regulated in glial cells by negative feedback mechanisms involving anti-inflammatory proteins such as IL-10 and by nuclear co-repressor proteins that inhibit transcription factors such as NF-κB/p65 from activing inflammatory gene promoters (Collingwood et al., 1999). Activation of inflammatory signaling pathways in glia during neurotoxic injury or neurodegenerative disease can produce pathologic levels of neurotoxic inflammatory mediators contributing to loss of neurons (Gao and Hong, 2008).

Nurr1 (NR4A2) tonically inhibits inflammatory gene expression in glial cells by stabilizing nuclear co-repressor proteins such as CoREST and NCoR2 at NF-κB/p65 binding sites (Saijo et al., 2009). When activated by sumolyation and phosphorylation, Nurr1 prevents expression of inflammatory genes, whereas selective knockdown of Nurr1 in glia exacerbates dopamine neuron loss in the SNpc after LPS treatment (Saijo et al., 2009). The capacity of Nurr1 to suppress inflammatory gene expression in glial cells suggests that this pathway could be viable target for inhibition of NF-κB-regulated neuroinflammatory genes in the CNS.

We recently reported that selected analogs 1,1(3′indolyl)-1-(p-subsitiutedphenyl)methanes (C-DIM) compounds, derived from 3,3′-diindolylmethane (DIM), a metabolite of the phytochemical indole-3-carbinol, decreased cytokine-induced expression of inducible nitric oxide synthase (iNOS/NOS2) in astrocytes by preventing DNA binding of NF-κB (Carbone et al., 2008; Tjalkens et al., 2008). Studies in cancer cells revealed that selected compounds in this series have specific activity toward the NR4A family of nuclear receptors, including NR4A1/TR3/Nur77 and NR4A2/Nurr1 (Inamoto et al., 2008; Li et al., 2012; Yoon et al., 2011). A para-methoxy substituted C-DIM, DIM-C-pPhOCH3 (C-DIM5), as well as DIM-C-pPhOH (C-DIM8), modulate Nur77 expression levels in pancreatic and colon cancer cells (Lee et al., 2010, 2014a; Yoon et al., 2011) the para-chloro-substituted analog, DIM-C-pPhCl (C-DIM12), is a high-affinity transactivator of Nurr1 in bladder cancer cells (Inamoto et al., 2008).

Previous pharmacokinetic and pharmacodynamic efficacy studies demonstrated that selected C-DIM compounds were protective against loss of dopamine neurons in the SNpc following lesioning with MPTP and probenecid (MPTPp; De Miranda et al., 2013). These studies also demonstrated that C-DIM5, C-DIM8 (DIM-C-pPhOH), and C-DIM12 cross the blood-brain barrier (BBB) and are orally bio-available (De Miranda et al., 2013). We therefore postulated that the neuroprotective effects of C-DIMs involve inhibition of glial activation and blockade of NF-κB-regulated neuroinflammatory genes, as well as through enhanced expression of Nurr1. To test this hypothesis, we dosed transgenic NF-κB/EGFP mice with MPTPp over 1 week and examined the capacity of selected C-DIM compounds to suppress ongoing loss of dopamine neuron and glial activation when administered after MPTPp. We report that selected C-DIM compounds prevented dopamine neuron loss in this model and attenuated activation of both microglia and astrocytes in the ventral midbrain. These results suggest that C-DIM compounds may exert their neuroprotective efficacy through transrepression of NF-κB-regulated inflammatory gene expression in glial cells and could be a potential new therapeutic modality for limiting neuronal loss during neurotoxic and neuroinflammatory injury.

MATERIALS AND METHODS

Chemicals and reagents

DIM-C-pPhOCH3 (C-DIM5), DIM-C-pPhOH (C-DIM8), and DIM-C-pPhCl (C-DIM12) were synthesized and characterized as described previously (Qin et al., 2004). MPTP and probenecid and all other general reagents were purchased from Sigma-Aldrich (St. Louis, MO).

MPTP and probenecid treatments

Transgenic NF-κB/EGFP reporter mice (C57Bl/6 background) were obtained as generous gift from Dr Christian Jobin (University of North Carolina, Chapel Hill) and described in (Magness et al., 2004). Dosing with MPTP and probenecid was conducted as described in De Miranda et al. (2013). Briefly, adult mice aged up to 12 weeks were randomly divided into treatment groups and administered probenecid (250 mg/kg, ip.) followed four h later by MPTP (20 mg/kg, subcutaneous) or saline every other day for 7 days (80 mg/kg MPTP total, 4 doses; Miller et al., 2011). After day 7, C-DIM compounds dissolved in corn oil (or corn oil vehicle control) were administered to mice once daily by oral gavage for 7 days. Mice were terminated on days 7 and 14; behavior was assessed at these timepoints and prior to treatment (day 0). Intrinsic NF-κB/EGFP tissue co-localization analysis (Figure 5G) was generated using saline or MPTP (80 mg/kg MPTP total, 4 doses over 14 days) and probenecid (250 mg/kg) with concurrent daily oral gavage (50 mg/kg) of C-DIM12 or vehicle control (corn oil). All animal procedures were performed in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Colorado State University Institutional Animal Care and Use Committee. Every effort was made to minimize pain and discomfort. Terminal procedures were performed under deep isoflurane anesthesia.

FIG. 5.

FIG. 5.

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C-DIM compounds modulate intrinsic expression of NF-κB/EGFP in the SN after MPTPp treatment. A–C, Representative tissue sections from the SN of transgenic NF-κB-EGFP mice show global increases in intrinsic GFP expression after MPTPp7d and MPTPp14d treatments expressed in pseudocolor, where red is most intense (20X montage, 40X inset, scale bar 100 µm). D–F, SN tissue sections from C-DIM-treated animals show a decrease in total NF-κB-EGFP expression at MPTPp14d. G, Co-localization of NF-κB-EGFP in TH+ neurons of the SN (40X) from animals treated with SAL or MPTPp14d concurrently given vehicle control or C-DIM12. H–I, Astrocyte (GFAP) and microglia (IBA-1) representative 40X images from the SN of MPTPp14d animals show co-localization of NF-κB-EGFP reporter (green), DAPI (blue). J, K, Scatter-plot analysis of GFAP (RhodDual) or IBA-1 (Cy5) and NF-κB (FITC) channels, show a more intense overlap of NF-κB-EGFP pixel intensity in microglia than astrocytes (SlideBook software co-localization analysis of pixel intensity minus background; F/F0).

Immunohistochemistry

Tissue processing was performed as reported previously (Miller et al., 2011; De Miranda et al., 2013). At 7 and 14 days, animals were terminally anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde. After perfusion, brains were removed and immersion fixed in 4% paraformaldehyde at 4°C for 3 h. The brains were then transferred to cacodylate-phosphate-buffered saline containing 15% sucrose overnight, followed by 30% sucrose. The tissue was then frozen in OCT and sectioned at 40 μm thickness on a microtome. Sections were stored at −20°C, free floating in cryoprotectant (30% w/v sucrose, 30% v/v ethylene glycol; 0.5 M phosphate buffer; pH 7.2) until staining.

Immunoflouresence tissue staining was completed as described in Miller et al. (2011). Antibodies were diluted in TBS and blocking buffer for a final concentration of 1:500; GFAP, Nitrotyrosine (3-NTyR; Cell Signaling, Danvers, MA), IBA-1 (Wako Chemicals USA, Irvine, CA), γH2Ax, MAP2, Tyrosine Hydroxylase (Abcam, Cambridge, MA), Nurr1 (Santa Cruz Biotechnology, Santa Cruz, CA).

Stereological counting of dopamine neurons

As reported in Miller et al. (2011), free-floating serial sections (40 µm) used for tyrosine hydroxylase (TH; ab152 Millipore, Billerica, MA) staining were obtained using systematic sampling from all sections encompassing the entire length of the SNpc (rostal boundary was marked by the subthalamic nucleus; caudal boundary was marked by the retrorubal field; Baquet et al., 2009). Every third tissue was selected and counted, for a total of 20 sections per animal. Stereological counts of TH-positive cells were performed using Slidebook software (version 5.0; Intelligent Imaging Innovations, Denver, CO) using the optical fractionator method (West and Gunderson, 1990). Images were captured using a Zeiss Axiovert 200M inverted fluorescence microscope equipped with a Hammatsu ORCA-ER-cooled charge-coupled device camera (Hammamatsu Pho- tonics, Hamamatsu City, Japan). The boundary of the SNpc was determined using low-magnification (×10) montage imaging. Total numbers of TH-positive cells were obtained through imaging (40x) uniform randomly placed counting frames (100 × 100 mm) with use of an optical dissector of 30 mm with 5 mm upper and lower guard zones. Representative montage images were generated for each treatment group with use of a 20x objective. Anatomic landmarks were used to select striatal sections for TH intensity staining in an identical process as described above and in Miller et al. (2011), staining all treatment groups simultaneously. Montage images of the ST were acquired using a 10X air Plan Aprochromat objective and masked to generate an outline of the striatum, where mean fluorescence intensity was subsequently analyzed. Representative high resolution montage images were generated for each treatment group with use of a 20X air Plan Aprochromat objective.

Total neuron counts and assessment of dopamine neuron damage

Three tissue sections (SN) per animal were selected for MAP2/TH co-immunofluorescence staining with DAPI counterstain; Anti-Tyrosine Hydroxylase antibody (ab76442, Abcam). Tissue sections were selected using similar anatomical landmarks, encompassing the rostral, medial, and caudal portions of tissue expressing TH+ neurons as assessed by stereology. The selected sections were imaged using the same optical fractionator method and counting frame boundaries as described in stereology methods, above. MAP2 positive neurons are reported as an average of raw MAP2 neuron counts per treatment group (N = 3 animals per group). Additional SN serial sections (3 per animal) from each treatment group were examined from the same anatomical landmarks within the boundary of the SN for assessment of 3-NTyR and γH2Ax co-expression with TH and DAPI counterstain. High-resolution (40X) images of TH-3NTyR were assessed for mean fluorescence intensity of 3-NTyR in TH-positive neurons and were quantified using mean intensity measurements normalized using background subtraction function of Slidebook software (N = 5). γH2Ax nuclear foci quantitation is expressed as the mean intensity of γH2Ax within the nucleus of TH+ neurons, normalized using background subtraction (N = 5 animals per group). All counts and assessments were coded for blind analysis.

Assessment of gliosis

Three sections from the SN (using stereological boundaries) and ST were selected based upon similar anatomical landmarks across treatment groups for co-immunofluorescence expression of GFAP and IBA-1 with DAPI counterstain. Sections were imaged at low magnification (×10) to identify the anatomical boundary of the SN and ST followed by high-resolution (40X) Z-stack images, flattened to maximum projection for blind counting assessment within each 40X counting frame. Counting frames were generated using the same guard distance and parameters of stereological counts GFAP and IBA-1 raw counts per 40X counting frame were averaged across treatment groups and reported as mean number of cells counted per frame (N = 6 animals per group).

Nurr1 expression and quantitation

Anatomically similar serial sections of the SN within the stereological count boundaries were selected for Nurr1 and TH co-immunofluorescence with DAPI counterstain (Nurr1 Antibody N20; Santa Cruz Biotechnology, Santa Cruz, CA). High-resolution (63X) images of TH-Nurr1 were assessed for mean fluorescence intensity of Nurr1 within TH-positive neurons and were quantified using mean intensity statistics, normalized using the background subtraction function of Slidebook software. Additional fluorescence intensity of total Nurr1 protein was measured by creating a segment on Nurr1, while removing pixels <10 μm (N = 4 animals per treatment group).

Behavioral analysis

Mice were evaluated at days 0, 7, and 14 for changes in locomotor function using VersaMax open-field behavior chambers (Accuscan Instruments, Inc., Columbus, OH). Mice were monitored over a 10-min period, under lowered ambient light and white noise. VersaDat software (Accusan Instruments, Inc.) was used to analyze open field behavior parameters. Hind-limb stride length measurements were conducted using an adapted methodology developed by Miller et al. (2011). Stride length was measured 1 day prior to treatment (day 0), on day 7 after MPTPp treatment, and day 14. Freely moving mice were trained to walk across a plexiglass trackway (5 cm × 1 m) and paw placement was recorded using a digital video camera. Stride length was calculated using Tracker Video Modeling (Tracker v.4.85 for MacOS X). Initial paw steps as well as those made immediately adjacent to the cage opening were omitted. Data are reported as average stride length (mm) per treatment group at the endpoint of the study, 7 or 14 days.

Immunoblotting

Striatal brain tissue was isolated from all treatment groups as described by De Miranda et al. (2013). Briefly, mice were decapitated under deep isoflurane anesthesia prior to rapid dissection of the brain into SN (for RNA analysis, see below) and striatal sections, which were snap-frozen using liquid nitrogen followed by storage at −80°C. Striatal tissue was homogenized using a Dounce tissue grinder and glass pestle, isolated in RIPA buffer with protease inhibitor. Protein was quantified using a BCA protein assay (Thermo Scientific Pierce, Rockford, Il) and equal protein (20 μg) was loaded in a polyacrylamide 12% gel with 4% stacking gradient. TH (AB152, Millipore), DAT (AB1591P, Millipore), and VMAT2 (AB15314, SantaCruz) protein quantitation were imaged using a Biorad ChemiDoc XRS System and compared with β-actin control using ImageJ analysis software (Schneider et al., 2012).

RNA isolation and real-time polymerase chain reaction array analysis of NF-κB-regulated inflammatory genes

Frozen sections of SN were processed for RNA isolation using the QIAgen RNeasy kit (QIAGEN, Valencia, CA) as per the manufacturer’s protocol, with the addition of both on-column and in-solution DNase treatments. Expression of NF-κB-regulated inflammatory genes from the SN of each treatment group was analyzed using a 384-well plate-based quantitative polymerase chain reaction (qPCR) array (PAMM-025Z, SA Biosciences, Frederick, MD) for 86 genes regulated by the NF-κB signaling pathway. cDNA was synthesized using BioRad iScript (CA) and real-time PCR Array analysis was completed using the PCR LightCycler489 PCR system (Roche Applied Sciences, Indianapolis, IN). Changes in gene expression were calculated based upon Ct values normalized to the internal standard reference genes β-actin, gapdh, β-2 microglobulin and heat shock protein 90 (HSP90ab1).

Statistical analysis

Data are presented as mean ± SEM. Experimental group analyses were preformed using a one-way analysis of variance (ANOVA) with a Tukey post hoc test. A Grubbs’ test (α = 0.05) was performed to identify significant outliers of treatment groups. Statistical significance was considered at *P < .05, **P < .01, ***P < .001, ****P < .0001 unless described additionally in figures. All statistical analyses were completed using Prism (version 6.0; Graph Pad Software, San Diego, CA).

RESULTS

C-DIM Compounds Prevent Loss of Dopamine Neurons in the SN After Lesioning With MPTP and Probenecid

To induce progressive loss of DA neurons in the SN, adult transgenic NF-κB/EGFP mice were treated with MPTPp (20 mg/kg MPTP, 250 mg/kg probenecid), every other day for 7 days in a repeated study as previously described by De Miranda et al. (2013). Stereological counting of TH-positive cells in the SNpc after the first 7 days of dosing indicated a loss of ∼50% of dopamine neurons (Figure 1A, B, and H). Quantification of immunofluorecence staining for TH in the striatum indicated a loss of dopaminergic terminals after 7 days’ treatment with MPTPp (Figure 1A, B, and I). Beginning on day 7, mice were administered C-DIM compounds (C-DIM5, C-DIM8, or C-DIM12, 50 mg/Kg) or corn oil vehicle control once daily by oral gavage. Mice that received only corn oil (MPTPp14d) exhibited ∼25% further dopamine neuron loss of DA neurons by day 14, as well as a further reduction in striatal TH intensity (Figure 1C and I). In contrast, mice receiving C-DIM5, C-DIM8, or C-DIM12 displayed no significant difference in dopamine neuron number or ST terminal intensity from MPTPp7d animals (Figure 1D, E, H, and I). These data demonstrate that MPTPp-induced neurodegeneration using this treatment regimen resulted in loss of TH-positive neurons in the SN over 7 days that continued to progress from days 7 to 14, even after cessation of treatment. Administration of C-DIM compounds beginning on day 7 effectively prevented any further loss of TH-positve neurons in the SNpc or terminals in the ST.

FIG. 1.

FIG. 1.

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Progressive dopamine neuron loss by MPTPp is attenuated by C-DIMs. A, Dosing regimen and timeline schematic for MPTP (total, 80 mg/kg) and probenecid (250 mg/kg) treatment in adult NF-κB-EGFP reporter mice, followed by tissue analysis. B–D, Representative images of TH-positive neuronal cell bodies in the SN and nerve terminals in the ST that are significantly reduced after 7 days of MPTPp treatment (MPTPp7d) and continue to degenerate 7 days after neurotoxin is removed (MPTPp14d; 20X montage images, scale bar 100 µm). E–G, Representative images of C-DIM5, C-DIM8, and C-DIM12 daily oral gavage (50 mg/kg) beginning at MPTPp7d attenuates dopamine neuron loss at MPTPp14d (corn oil daily gavage, vehicle control). H, Stereological counts of TH-positive neurons in the SNpc are significantly reduced at MPTPp7d worsening at MPTPp14d compared with saline. TH-positive neurons are significantly protected in C-DIM-treated animals compared with their MPTPp14d counterparts. I, Mean fluorescence intensity of TH in the ST is significantly reduced in MPTPp7d, progressing to further loss at MPTPp14d, whereas C-DIM treatments prevented additional loss. Data are expressed as mean ± SEM (N = 6); horizontal bars represent statistical significance between related groups. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Dopamine Terminal Integrity and Neurobehavioral Analysis

Levels of several proteins expressed in dopamine neuron terminals in the striatum were examined by immunoblotting in MPTPp14d mice receiving either C-DIM compounds or corn oil vehicle control. Analysis of TH, DAT, and VMAT2 revealed a significant reduction in TH and DAT in MPTPp7d mice that was further reduced in MPTPp14d animals (Figure 2C and E). Mice that received daily oral gavage with C-DIM5, C-DIM8, or C-DIM12 had significantly increased levels of TH and DAT compared with mice that received only corn oil (MPTPp14d). Expression of VMAT2 followed the same trend, although quantitative analysis did not indicate significant changes across treatment groups. The capacity of C-DIM compounds to increase protein expression of TH, DAT, and VMAT2 in the striatum paralleled the survival of dopaminergic cell bodies in the SN, as well as the evident preservation of TH+ dopaminergic terminals in the striatum.

FIG. 2.

FIG. 2.

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Dopamine terminal proteins and locomotor behavior after MPTPp and C-DIM treatment. A, Stride length measured in mice at MPTPp14d is significantly reduced from saline, and improved with C-DIM treatment. B, Open field behavior (rearing movement #) did not reveal changes between treatment groups (N = 6). C–E Western blots of TH, DAT, and VMAT proteins from the striatum show lower expression at MPTPp7d, worsening at MPTPp14d, as compared with β-actin; relative optical density compares quantitative blot measurements (N = 4); #P < .1, 90% confidence interval. Data are expressed as mean ± SEM; horizontal bars represent statistical significance between related groups. *P < .05, **P < .01.

Locomotor function was evaluated to determine differences between animals receiving C-DIMs or corn oil after onset of dopamine neuron loss. Analysis of stride length indicated a significant reduction in locomotor function in MPTPp14d mice that was prevented by treatment with C-DIM5 and C-DIM12 (Figure 2A). C-DIM8-treated mice demonstrated a trend toward reversal of MPTPp-induced decreases in stride length that was not significant at this timepoint. Measures of locomotor function using open field behavioral analysis, such as rearing movement and total distance traveled (data not shown), did not show detectable differences between treatment groups (Figure 2B).

C-DIM Compounds Reverse the Effects of MPTPp on Neuronal Injury in the Substantia Nigra

To determine changes in the total number of neurons in the SNpc, we used immunofluorescence to concurrently label TH-positive neurons, as well as neurons expressing microtubule-associated protein-2 (MAP2). Analysis of MAP2 and TH expression immunofluorescence microscopy (Figure 3A) demonstrated co-localization of both epitopes in neuronal cell bodies in the SNpc, as well as neurons expressing only MAP-2 (montage and high magnification inset). There was a significant loss of MAP2+ neuronal cell bodies in the SN of animals treated with MPTPp at day 14 (Figure 3B). This neuronal loss was supported by morphological examination of stained brain tissue in the MPTPp14d group, which indicated a consistent absence of MAP+ neurons in all animals examined (n = 6). The number of MAP2-positive neurons in C-DIM-treated mice at day 14 was not statistically different from the MPTPp7d group or the MPTPp14d group, despite a clear trend toward increasing numbers of neurons by day 14 (Figure 3B).

FIG. 3.

FIG. 3.

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C-DIM compounds prevent neuronal death and dysfunction after MPTPp. A, Representative 20X images of TH (green), MAP2 (red), and DAPI (cyan) show total neuron staining compared with TH-positive immunofluorescence, (63X inset). B, Quantitative MAP2 neuron counts of total neuron loss, significantly reduced at MPTPp14d, attenuated with C-DIM treatment (N = 3). C–H Representative 40X images of nitrotyrosine adducts on TH neurons, identified by 3-Nitrotyrosine immunofluorescence (yellow), co-localizes with TH-positive neurons (blue), DAPI (cyan), increases with MPTPp treatment, but is reduced with C-DIMs. I, Quantitative measurement of nitrotyrosine mean fluorescence intensity within TH-positive neurons shows significant increases at MPTPp7d and MPTPp14d, reduced by C-DIM treatment. (N = 5). J–O, Representative 40X images of apoptosis marker γH2AX (green) increases in TH-positive neurons (red), DAPI (cyan), at MPTPp7d and MPTPp14d. P, The mean intensity of γH2AX in TH+ neurons (nuclear foci) is significantly increased in MPTPp14d-treated animals, but is reduced by C-DIM treatment (N = 5). Data are expressed as mean ± SEM; horizontal bars represent statistical significance between related groups. *P < .05, **P < .01, ***P < .001.

Activated glial cells produce high levels of nitric oxide (NO) that forms peroxynitrite (ONOO−) under conditions of oxidative stress (Brambilla et al., 2005), which is damaging to neuronal mitochondria and leads to loss of dopamine synthesis, neuronal dysfunction, and activation of apoptotic signaling pathways (Crowe et al., 2011). Using 3-nitrotyrosine (3-NTyr) as a marker for nitrosative stress and peroxynitrite protein adducts in neurons, we found that mice exposed to MPTPp exhibited increased immunostaining for 3-NTyr adducts that co-localized with TH+ neurons (Figure 3C–E), whereas little to no 3-NTyr staining was detected in control mice. Mice administered C-DIM5, C-DIM8, or C-DIM12 after treatment with MPTPp had dramatically reduced staining for 3-NTyr adducts compared with MPTPp14d mice given only corn oil (Figure 3F–H). Quantitative measurements of 3-NTyr adducts by immunofluorescence in TH neurons revealed increased nitrosative stress in MPTPp7d and MPTPp14d animals that was prevented by treatment with C-DIM5 and C-DIM12, although only partially decreased by C-DIM8 (Figure 3I).

Apoptosis in TH neurons in the SN was identified by the presence of phosphorylated histone H2A.X (anti-γH2AX), a sensitive marker of DNA double-stranded breaks (DSBs). Expression of γH2AX was increased in nuclei of TH+ neurons in MPTPp7d and MPTPp14d animals (Figure 3J–L). In contrast, mice that received C-DIM compounds displayed an absence of nuclear γH2AX staining that was comparable with control animals and less than either the MPTPp7d or MPTPp14d groups (Figure 3M–O). Co-localization of γH2AX with TH neurons in MPTPp14d mice indicates that these cells were undergoing DNA fragmentation and apoptosis following treatment with MPTPp that was prevented by concurrent treatment with C-DIM compounds.

C-DIM Compounds Prevent MPTPp-Induced Changes in Nurr1 Expression and Subcellular Localization in Dopaminergic Neurons

Nurr1 is an immediate-early response protein highly expressed in dopaminergic brain regions (Maxwell and Muscat, 2006; Zetterström et al., 1997). Nurr1 also has both nuclear import and export peptide sequences (NLS/NES), which mediate export of the protein from the nucleus to the cytoplasm under oxidative conditions, suggesting that Nurr1 is functionally regulated by its subcellular localization (García-Yagüe et al., 2013). It is unclear whether this same phenomenon occurs in dopamine neurons in vivo following exposure to MPTPp. We therefore compared the expression and subcellular localization of Nurr1 in the SNpc between control, MPTPp14d and MPTPp14d + C-DIM mice using immunofluorescence to identify direct modulatory effects of C-DIM compounds on the expression and cellular morphology of Nurr1 in dopaminergic neurons (Figure 4). In the SNpc of saline-treated control mice, Nurr1 was localized primarily to the nucleus of TH+ neurons (Figure 4A). In contrast, expression of Nurr1 in TH+ neurons in the MPTPp7d group was almost exclusively cytoplasmic, indicating translocation and export of Nurr1 from the nucleus subsequent to MPTP-induced oxidative and inflammatory stress (Figure 4B). Expression of Nurr1 in the MPTPp14d group was also primarily evident in the cytoplasm, although its overall expression steadily decreased from days 7 to 14 after the initiation of treatment with MPTPp (Figure 4C). In MPTPp14d animals receiving C-DIM compounds, MPTPp-mediated downregulation of Nurr1 expression was inhibited in both TH+ dopaminergic neurons (Figure 4F) and throughout the SNpc (Figure 4G). Moreover, in the MPTPp14d + C-DIM12 group, there was a striking preservation of nuclear Nurr1 expression at 14 days (Figure 4D) that further increased in overall intensity after 21 days (Figure 4E–G). Quantitative determination of Nurr1 expression 21 days following the onset of MPTPp treatment in mice given corn oil vehicle (MPTPp21d) or C-DIM12 (MPTPp21d + C-DIM12) indicated that expression of Nurr1 continues to decline in the SNpc even 2 weeks after cessation of treatment with MPTPp but is markedly preserved by daily oral treatment with C-DIM12 (Figure 4F and G).

FIG. 4.

FIG. 4.

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Nurr1 translocation and expression in dopamine neurons. A–C, Representative 63X images of tyrosine hydroxylase positive neurons (TH; red) and Nurr1 (green) from 40-µm sections of the SN show cytoplasmic translocation of Nurr1 following MPTPp treatment. D–E, Post-lesion treatment with C-DIM12 shows increases in Nurr1 expression in dopamine neurons, where the greatest measured nuclear translocation occurred at MPTPp21d. F–G, Mean fluorescence intensity of Nurr1 in TH-positive neurons and total Nurr1 expression were quantified using mean intensity measurements normalized using background subtraction (Slidebook software co-localization analysis of F/F0). Data are expressed as mean ± SEM (N = 3); horizontal bars represent statistical significance between related groups. *P < .05.

MPTPp-Induced Expression of NF-κB/EGFP in the Substantia Nigra is Reduced by C-DIM Compounds

Chronic activation of glial cells is closely linked to progressive dopamine neuron death in PD through increased expression of pro-inflammatory factors regulated by the transcription factor, NF-κB. We therefore used transgenic reporter mice that express EGFP upon binding of p65/p50 to high-affinity NF-κB promoter elements (Magness et al., 2004) to identify functional changes in NF-κB activation during exposure to MPTPp in the absence or presence of C-DIM compounds. Coronal sections within the boundary of the SN from each treatment group were assessed for total intrinsic GFP expression by fluorescence micrscopy at 490 nm excitation/520 nm emission and images were rendered in pseudocolor to highlight intensity of the EGFP reporter (Figure 5A–E). Basal expression of NF-κB/EGFP in the saline-treated control group (Figure 5A) was notably increased in MPTPp7d animals and remained high in the MPTPp14d group (Figure 5B and C). C-DIM5, C-DIM8, and C-DIM12 globally decreased expression of NF-κB/EGFP to levels below the MPTPp7d threshold similar to those observed in control mice (Figure 5D–F). High magnification images (40X insets) of the SN indicate that compared with glial cells, neurons expressed greater levels of intrinsic GFP, likely due to their high constitutive activity of the NF-κB pathway compared with glial cells. Intrinsic NF-κB reporter fluorescence was confirmed present in TH+ neurons in animals treated with SAL or MPTPp14d and concurrent oral gavage with vehicle control (corn oil) or C-DIM12 (G). In order to determine expression of NF-κB/EGFP in glial cells following treatment with MPTPp, sections from the SN were examined by immunofluorescence microscopy for co-expression of NF-κB/EGFP in astrocytes (GFAP) and microglia (IBA-1). Co-expression of intrinsic GFP with either GFAP or IBA-1 in MPTPp14d tissue (Figure G and H) indicated that activated astrocytes and microglia both expressed high levels of EGFP in areas with active neurodegeneration in the SNpc that was undetectable in C-DIM-treated animals (data not shown for brevity). Representative 40X images of astrocytes (Figure 5H) and microglia (Figure 5I) show that both cell types expressed the NF-κB/EGFP reporter in MPTPp14d mice. Scatter plots of fluorescence channel intensity (Figure 5J and K) indicated that IBA-1 intensity more closely overlapped with intrinsic GFP intensity than did GFAP intensity, suggesting that NF-κB/EGFP was more highly activated in microglia than in astrocytes in the MPTPp14d treatment group.

Glial Activation in the Midbrain is Suppressed by Treatment with C-DIM Compounds

To examine the sequence and severity of inflammatory glial activation following treatment with MPTPp, we measured the relative activation of astrocytes and microglia in the SN and the ST based upon expression of GFAP and IBA-1 (Figure 6). In montage images of stained coronal sections of the SN, treatment with MPTPp increased expression of both GFAP (green) and IBA- (red) throughout the SNpc and SNr from 7 to 14 days after the initiation of exposure (Figure 6A–C), paralleling the progression of dopamine neuron loss. IBA-1 expression in the SN was more extensive in MPTPp14d mice compared with MPTPp7d animals, whereas GFAP levels remained approximately constant between MPTPp7d and MPTPp14d mice (Figure 6A–C). High magnification images of microglia and astrocyte morphology in the SN revealed that in tissue from saline-treated control animals, microglia expressed a ramified morphology consistent with a resting phenotype (Figure 6G) but were markedly activated in MPTPp14d tissue, exhibiting a more amoeboid shape and surrounding cells with the appearance of nuclear condensation (Figure 6H). Astrocytes also displayed a hypertrophic and activated phenotype in MPTPp14d animals. The morphology of both microglia and astrocytes was markedly less activated in C-DIM-treated animals, phenotypically resembling those in the control group (Figure 6I). Quantitative counts of GFAP- and IBA-1-positive cells in the SN and ST indicated a large increase in the number of activated glial cells in MPTPp7d and MPTPp14d mice over saline-treated controls (Figure 6J–M). In the SN, the number of IBA-1-postive cells appeared to increase slightly in the MPTPp14d group compared with the MPTPp7d group (Figure 6K), whereas the number of GFAP-positive cells remained constant from days 7 to 14 (Figure 6J). In the ST, there was a sustained elevation in the number of GFAP+ and IBA-1+ cells in the MPTPp7d and MPTPp14d groups (Figur 6L and M). In both the SN and ST, treatment with C-DIM compounds reduced the number of activated glial cells compared with the MPTPp7d and MPTPp14d groups (Figure 6J–M).

FIG. 6.

FIG. 6.

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Activation of microglia and astrocytes in the SN during progressive dopamine neuron loss is prevented by C-DIM compounds. A–C, Representative immunofluorescence 20X montage images in the SN of astrocyte (GFAP, green) and microglia (IBA-1, red) activation at MPTPp7d and MPTPp14d compared with saline. D, E, Astrocyte and microglia activation in the SN of C-DIM5, C-DIM8, or C-DIM12 at MPTPp14d. G, Astrocyte (GFAP, green) and microglia (IBA-1, red) morphology in the SN of saline tissue show a resting phenotype. H, Activated astrocytes in MPTPp14d express increased GFAP; activated microglia undergo morphological changes consistent with a phagocytic phenotype, including retracted processes and an amoeboid shape (arrows) capable of engulfing debris. I, Treatment with C-DIM12 at MPTPp7d reduces microglial activation at MPTPp14d; microglia express a less-activated phenotype. J-M. Quantitative counts of GFAP and IBA-1 positive cells in the SN and ST show increased gliosis at MPTPp7d and MPTPp14d, reduced by C-DIM treatment. Data are expressed as mean ± SEM (N = 6); horizontal bars represent statistical significance between related groups. *P < .05, **P < .01, ***P < .001, ****P < .0001.

Expression of NF-κB Regulated Inflammatory Genes is Reduced in C-DIM-Treated Mice

To determine the effect of C-DIM compounds on expression of NF-κB-regulated neuroinflammatory genes in MPTPp-treated mice, we analyzed mRNA isolated from the SN of animals given saline, MPTPp14d, MPTPp14d + C-DIM5, and MPTPp14d + C-DIM12 treatments using real-time qPCR arrays (qRT arrays) specific for the NF-κB signaling pathway (Figure 7). C-DIM5 and C-DIM12 selected as representative activators of NR4A1/Nur77 and NR4A2/Nurr1, respectively. The qRT arrays used contain 86 different NF-κB-regulated genes-related t-cell division, apoptosis, and inflammation (SA Biosciences, Gaithersburg, MD). Most of the genes that were significantly increased in MPTPp14d animals over saline controls were inflammatory cytokines, chemokines, and receptors (representative genes, Figure 7A–H). Animals that received C-DIM12 daily by oral gavage expressed significantly decreased levels of cytokines and chemokines (TNFα, IL-1α, and CCL2) and mediators of inflammation (Caspase 1, TLR4). Interestingly, animals that received C-DIM5 did not display as significantly reduced levels of inflammatory gene expression at MPTPp14d, although a decreasing trend was evident (n = 4 separate animals). The fold-difference in expression of IL-10, IFNγ, and Nod1 (Figure 7J–L) also demonstrated that at MPTPp14d, proinflammatory gene expression was increased above control values. In contrast, C-DIM5- and C-DIM12-treated animals displayed levels of IFNγ and Nod1 mRNA below that of control animals. Considered an anti-inflammatory cytokine, IL-10 levels in MPTPp14d mice were downregulated relative to control in MPTPp14d mice (Figure 7J) but in C-DIM treated animals, levels of IL-10 mRNA trended upward, indicating a possible role for this cytokine C-DIM mediated suppression of neuroinflammation.

FIG. 7.

FIG. 7.

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NF-κB-regulated inflammatory gene expression in the SN. RNA isolated from the SN of Saline, MPTPp14d, and C-DIM5- or C-DIM12-treated animals was analyzed using a real-time polymerase chain reaction (PCR) array of NF-κB pathway genes (SA Biosciences). A-H, Average ΔCt values from real-time analysis show global increases in NF-κB-regulated genes from saline to MPTPp14d (student’s t-test, #P < .1, 90% confidence interval). J–L, Fold regulation over saline of IL-10 shows a trend for increased expression in C-DIM5 and C-DIM12 over MPTPp14d, whereas INFγ and Nod1 are increased in MPTPp14d but reduced in C-DIM5 and C-DIM12 treatments. Data are expressed as mean ± SEM (N = 4); horizontal bars represent statistical significance between related groups. *P < .05, **P < .01.

DISCUSSION

Neuroinflammation remains an intractable therapeutic problem (Glass et al., 2010; Maguire-Zeiss and Federoff, 2010). Potential disease modifying agents have often failed in phase II and III clinical trials, typically for lack of efficacy (Gao and Hong, 2008; Glass et al., 2010). The MPTPp model of PD described here is useful for examining neuroinflammation, because loss of dopamine neurons in the SNpc continues even after cessation of treatment with MPTPp and is associated with marked inflammatory activation of microglia and astrocytes. The ability of C-DIM compounds to reduce both NF-κB/EGFP reporter activity as well as the expression of NF-κB-regulated inflammatory genes in the SN suggests a direct effect on suppression of neuroinflammatory signaling in glial cells that is protective towards dopaminergic neurons.

Treatment with MPTPp in this model caused progressive loss of dopamine neurons in the SNpc and not merely phenotypic decreases in expression of TH, which is important in determining neuroprotective efficacy (Carta et al., 2011; Petroske et al., 2001). Counterstaining for Nissl substance in conjunction with immunohistochemical (IHC) staining for TH is frequently used to compare total neuron survival to TH expression in the SN of MPTP-treated mice (Baiguera et al., 2012; Carta et al., 2011) but the use of transgenic fluorescent reporter mice in this study necessitated frozen sectioning to maintain activity of EGFP in conjuction with co-immunofluorescence with TH and MAP2, which is a more reliable marker using this technique than NeuN (Cannon and Greenamyre, 2009). Morphological and quantitative examination of neurons revealed that C-DIM treatment prevented loss of MAP-2+ neurons (Figure 3A and B) in addition to TH+ neurons (Figure 1), indicating that C-DIM compounds were neuroprotective against MPTP. The data in Figure 3 (J–O) demonstrating that C-DIM compounds decrease the number of histone γ-H2AX+ cells undergoing apoptosis also confirms a reduction in neuronal death (Crowe et al., 2011). In seizure models, the appearance of γ-H2AX staining in neuronal nuclei following excitotoxic injury represents a threshold after which DNA repair mechanisms are overwhelmed and cells incur DNA damage (Crowe et al., 2011). Here, we observed apoptotic dopamine neurons with DNA DSBs in MPTPp7d mice that further increased in MPTPp14d mice but were prevented by treatment with C-DIM compounds (Figure 3J–O). These results suggest that C-DIM compounds reduce DNA damage in the SN to levels commensurate with cellular repair mechanisms.

C-DIM treatment maintained expression of TH and DAT in the striatum (Figure 2C and D) and also improved neurobehavioral function, based upon measurement of hind limb stride length (Figure 2A). Rearing movements and other parameters of open-field analysis did not show any measurable difference between treatment groups (data not shown), suggesting the lesion is at the threshold for detection of early-phase motor symptoms (Figure 2B). In humans, motor symptoms are clinically apparent at 70–80% dopamine loss from the ST, corresponding to ∼50–60% loss of dopamine neurons from the SN (Fearnley and Lees, 1991). Although severe ablation of the SN with MPTP is similar to end-stage PD (Petroske et al., 2001), the degree of neuron loss observed after only 4 doses of MPTPp in this model more closely mimics early motor deficits. These data suggest that C-DIM compounds are able to protect against this type of MPTPp-induced lesion within in the midbrain; however, the therapeutic efficacy of the C-DIM compounds will need to be assessed in additional models of PD. Chronic MPTPp studies are currently ongoing to examine the long-term efficacy of C-DIM12 in preventing loss of dopaminergic neurons concurrent with a neurotoxic insult.

The neuroprotective effects of C-DIM compounds also correlated with increased expression and nuclear localization of Nurr1 (Figure 4). This gene family includes the orphan receptors NR4A1 (Nur77/TR3), NR4A2 (Nurr1), and NR4A3 (Nor1; Mohan et al., 2012). Nurr1 is highly expressed in the basal midbrain during development and is required for maturation and homeostasis of dopamine neurons (Hermanson et al., 2003; Kadkhodaei et al., 2009). Individuals polymorphic for Nurr1 are at increased risk for developing late-onset PD (Chu et al., 2006; Le et al., 2008). As C-DIM12 and related analogs activate Nurr1 (Inamoto et al., 2008; Li et al., 2012), we investigated whether this compound had a direct effect on the subcellular localization of Nurr1 in dopamine neurons MPTPp-treated mice. A striking translocation of Nurr1 from the nucleus to the cytoplasm occurred in TH+ neurons in the SNpc following treatment with MPTPp (Figure 4A–E), paralleling increases in neuronal apoptosis and 3-NTyR protein adducts. C-DIM treatment prevented this translocation and increased expression of Nurr1 protein in TH+ and MAP-2+ neurons, particularly C-DIM12, which further increased Nurr1 expression as long as 14 days after the end of MPTPp treatments (MPTPp21d group, Figure 4F and G). Translocation of Nurr1 from the nucleus to the cytoplasm was also reported following treatment of MN9D neurons with the oxidizing agent sodium arsenite via exposure of a nuclear export signal (García-Yagüe et al., 2013). Enhanced expression and nuclear localization of Nurr1 is therefore likely to have significant neurotrophic effects by supporting a dopaminergic phenotype and maintaining function of neurons within the SN.

Activation of microglia and astrocytes results in the production of reactive oxygen and nitrogen species, as well as inflammatory cytokines and chemokines (Davalos et al., 2005; Glass et al., 2010; Hirsch and Hunot, 2009). Gliosis in the SN following MPTPp treatment (Figure 6) was characterized by hypertrophic changes in both astrocytes and microglia, with microglia exhibiting an amoeboid morphology surrounding fragmenting neuronal nuclei, characteristic of phagocytic activity (Brown and Neher, 2010; Polazzi et al., 2010). C-DIMs reduced these inflammatory changes and caused a decrease in NF-κB reporter activity in the SN, suggesting an inhibition of NF-κB-dependent inflammatory signaling in glial cells that reduced neuronal stress response pathways. The localization of NF-κB-EGFP expression within the SN appears to be most intense in the reticulata region, which is consistent with the pattern of gliosis observed in Figure 6. Neurons receiving inputs from the SNpc likely become stressed, resulting in activation of NF-κB in both these cells and in surrounding activated glial cells. The SN is primed for glial activation due to the large number of microglia (Lawson et al., 1990), as is evident from IBA-1 staining in the SNpc and SNr in control animals (Figure 6A), which become activated following MPTPp treatment.

Nurr1 constitutively inhibits inflammatory gene expression in glial cells by recruiting co-repressor complexes (CoREST, NCoR2, and HDAC) to NF-κB/p65 binding sites in inflammatory gene promoters (Saijo et al., 2009). Mice heterozygous for Nurr1 are more susceptible to the neurotoxic effects of NO following methamphetamine challenge and show increased apoptosis in dopaminergic neurons (Imam et al., 2005). Glial-derived NO is converted to ONOO− and forms 3-NTyr protein adducts in neurons that damage mitochondria (Liberatore et al., 1999). The capacity of C-DIM compounds to block 3-NTyr protein adduct formation within dopamine neurons (Figure 3C–I) is indicative of an anti-inflammatory mechanism and reduced production of NO by activated glial cells. NOS2 is inducibly expressed by NF-κB in activated glial cells and the reduction in 3-Nyr-labeled dopamine neurons in C-DIM-treated animals is consistent with the reduced expression of NF-κB-regulated inflammatory genes measured in qRT array studies (Figure 7).

The pattern of inflammatory gene expression observed here is consistent with human PD, where similar cytokines have been measured in blood and CSF (Hartmann et al., 2003). C-DIM5 and C-DIM12 activate Nur77 and Nurr1, respectively (Inamoto et al., 2008; Lee et al., 2011, Yoon et al., 2011) and decreased NF-κB-regulated genes, including IL-1α, CCL2 (Figure 7). The trend toward decreased expression of other genes (TNF, Caspase 1, and Card10) is likely related to the relatively early stage of the lesion and to pharmacokinetic differences between C-DIM5 and C-DIM12; C-DIM5 is more rapidly metabolized when dosed orally (Cmaxbrain, 378 ng/ml) versus C-DIM12 (Cmaxbrain, 1,173 ng/ml; P < .0005) (De Miranda et al., 2013), possibly reducing the effectiveness of C-DIM5. This variation in neuroprotection may also be due to pharmacodynamic differences between the target receptors of C-DIM5 and C-DIM12, which have shown preferential binding affinities for NR4A1 and NR4A2, respectively (Inamoto et al., 2009; Lee et al., 2014b; Li et al., 2012; Yoon et al., 2011). In addition, the high expression of Nurr1 in the SN as well as the downstream targets and molecular function of each member of the orphan nuclear receptor family likely influences the neuroprotective efficacy of C-DIM12 (higher affinity for NR4A2/Nurr1) compared with C-DIM5 and C-DIM8 (higher affinity for NR4A1/Nur77).

CONCLUSIONS

The orphan nuclear receptor NR4A2/Nurr1 not only supports neuronal function but also modulates the activation of microglia and astrocytes in the adult brain, suggesting that it may play a dual protective role in CNS homeostasis. We demonstrated that selected activators of NR4A1/Nur77 and NR4A2/Nurr1 confer protection against loss of dopaminergic neurons following treatment with MPTPp in NF-κB/EGFP reporter mice. Treatment with C-DIMs also suppressed gliosis and expression of NF-κB-regulated inflammatory genes and increased the expression of Nurr1 while maintaining its nuclear localization in dopaminergic neurons. Collectively, these data support the utility of selected C-DIM compounds as neuroprotective agents in PD.

FUNDING

Rapid Research Innovation [grant from the Michael J. Fox Foundation to R.B.T.]; the National Institute of Health [Grant number P30-E5023512] and [Grant number R01-ES021656].

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