Anti-inflammatory and neuroprotective effects of an orally active apocynin derivative in pre-clinical models of Parkinson’s disease

Eight-week-old male C57BL/6 mice weighing 24 to 28 g were housed in standard conditions: constant temperature (22 ± 1°C), humidity (relative, 30%) and a 12 h light/dark cycle. Use of the animals and protocol procedures were approved and supervised by the Institutional Animal Care and Use Committee (IACUC) at Iowa State University (Ames, IA, USA) To assess the neuroprotective effect of diapocynin, we first used low doses of diapocynin (100 and 150 mg/kg/day) via oral gavage, but these doses showed only a moderate effect in attenuating MPTP-induced neurochemical deficits. Therefore, we used a 300 mg/kg dose of diapocynin in the subacute MPTP model of PD for detailed characterization of the neuroprotective efficacy of diapocynin. This 300 mg/kg dose was chosen based on apocynin, which has been used at a similar dose range in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease mouse models [14].

In the subacute MPTP regimen, mice received 25 mg/kg/day MPTP-HCl in saline intraperitoneally for 5 consecutive days. Diapocynin was dissolved in 10% ethanol 1 day before the MPTP insult and the drug treatment continued for 11 days. Animals were subjected to measurements of inflammatory markers, neurotransmitter levels, behavioral changes and neuronal damage at various time points. Control mice received equivolumes of the vehicle solution.

In the post-treatment regimen, diapocynin was administered 3 days after the MPTP treatment. For chronic MPTP treatment, mice received 2 doses of MPTP (25 mg/kg/dose, s.c.) and 2 doses of probenecid (250 mg/kg/dose, i.p.) per week for 5 consecutive weeks. Mice received 3 doses of diapocynin (100 mg/kg/day) per week by oral gavage, and the drug treatment started 1 week before MPTP injections, continued throughout the MPTP injection period and extended for another 45 days of post-MPTP treatment. After all treatments, animals were subjected to behavioral, neurochemical and histological measurements.

Samples were prepared and quantified, as described previously [15, 16]. On the day of analysis, neurotransmitters from striatal tissues were extracted using an antioxidant extraction solution (0.1 M perchloric acid, containing 0.05% Na₂EDTA and 0.1% Na₂S₂O₅) and isoproterenol (as internal standard). Dopamine, 3,4-dihydroxyphenyl-acetic acid (DOPAC) and homovanillic acid (HVA) were separated isocratically by a reversed-phase column with a flow rate of 0.6 ml/min. An HPLC system (ESA, Inc, Bedford, MA, USA) with an automatic sampler equipped with refrigerated temperature control (Model 542; ESA, Inc) was used for these experiments.

Diapocynin from the striatum and SN was quantified using the Agilent 1100 Series LC/MS binary pump (Agilent, Santa Clara, CA, USA), PDA detector (UV diode array detector) and an autosampler. On the day of analysis, a 20 μl sample was passed through the 0.2 μm filter at the eluent flow rate of 0.25 ml/min. Negative-ion, atmospheric pressure chemical ionization was used at amplitude 1.5 volts, and manual MS/MS was done. The mobile phase used in LC/MS consisted of a gradient elution. Solvent A was 480:20:0.38 water:methanol:ammonium acetate (v/v/w) and solvent B was 20:480:0.38 water:methanol:ammonium acetate (v/v/w). The standards series were taken as 0.3 μg, 1.0 μg, 3.0 μg, 10 μg, and 30 μg. The actual molecular weight of diapocynin is 329.1 g/mol, but by breaking the molecule in MS/MS it becomes 313.9 g/mol by elimination of one methyl molecule. The retention time for the diapocynin peak was 1.9 minutes. Data were fit to a straight line by linear regression analysis using Quant analysis software (Agilent, Santa Clara, CA, USA).

Mice were sacrificed 4 or 7 days after MPTP treatment, and striatum and SN tissues were dissected. Brain lysates containing equal amounts of protein were loaded in each lane and separated in a 10 to 15% SDS-polyacrylamide electrophoresis gel, as described previously [15, 17]. The membranes were then incubated with primary antibodies (anti-TH (Chemicon, Temecula, CA, USA), anti-Iba-1 (Abcam, Cambridge, UK), anti-GFAP (Chemicon), anti-iNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-gp91phox (Abcam), anti-3NT (Chemicon) and anti-4-HNE (R&D Systems, Minneapolis, MN, USA)). Next, membranes were incubated with Alexa Fluor 680 goat anti-mouse or Alexa Fluor 680 donkey anti-goat (Invitrogen, Carlsbad, CA, USA) or IRDye 800 donkey anti-rabbit (Rockland, Gilbertsville, PA, USA) secondary antibodies. To confirm equal protein loading, blots were reprobed with a β-actin antibody (Sigma-Aldrich, St Louis, MO, USA) at 1:10000 dilution. Western blot images were captured with a LI-COR Odyssey machine (LI-COR, Lincoln, NE, USA). The Western blot bands were quantified using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).

One day after the last MPTP treatment, mice were perfused with 4% paraformaldehyde (PFA) and post-fixed with PFA and 30% sucrose, respectively. Next, fixed brains were cut into 30 μm sections and were incubated with primary antibodies (anti-Iba-1 (Abcam), anti-GFAP (Chemicon), anti-iNOS (Santa Cruz Biotechnology), anti-3NT (Chemicon), anti-gp91phox (Abcam), anti-TH (Chemicon) and anti-4HNE (R&D)) overnight at 4°C. Appropriate secondary antibodies (Alexa Fluor 488 or 594 or 555; Invitrogen) were used, followed by incubation with 10 μg/ml Hoechst 33342 (Invitrogen) for 5 minutes at room temperature to stain the nucleus. Sections were viewed under a Nikon inverted fluorescence microscope (Model TE-2000U; Nikon, Tokyo, Japan); images were captured with a SPOT digital camera (Diagnostic Instruments, Inc, Sterling Heights, MI, USA).

DAB immunostaining was performed in striatum and SN sections, as described previously [3, 18]. Briefly, 30 μm sections were incubated with either anti-TH antibody (Calbiochem, Billerica, MA, USA; rabbit anti-mouse, 1:1800 dilution) or anti-Iba-1 (Abcam; goat anti-mouse, 1:1000 dilution) or anti-GFAP (Chemicon; mouse anti-mouse, 1:1000 dilution) antibody, followed by incubation with biotinylated anti-rabbit or goat or mouse secondary antibody. Total numbers of tyrosine hydroxylase (TH)-positive neurons in SN were counted stereologically with Stereo Investigator software (MBF Bioscience, Williston, VT, USA), using an optical fractionator [16, 19].

On the day of staining, sections were incubated with anti-TH antibody (Chemicon), followed by Alexa Fluor 568 donkey anti-mouse (Invitrogen) secondary antibody. Then FJB staining was done on the same sections by the modified FJB stain protocol, including incubation in 0.06% potassium permanganate for 2 minutes and 0.0002% FJB stain for 5 minutes. Sections were viewed under a Nikon inverted fluorescence microscope (Model TE-2000U; Nikon) and images were captured with a SPOT digital camera (Diagnostic Instruments, Inc).

An automated device (Model RXYZCM-16; Accuscan, Columbus, OH, USA) was used to measure the spontaneous activity of mice. The activity chamber was 40 × 40 × 30.5 cm, made of clear Plexiglas and covered with a Plexiglas lid with holes for ventilation. Data were collected and analyzed by a VersaMax Analyzer (Model CDA-8; AccuScan). Before any treatment, mice were placed inside the infrared monitor for 10 minutes daily for 3 consecutive days to train them. Five days after the last MPTP injection, open field and rotarod experiments were conducted. Locomotor activities were recorded for 10 minute test sessions. A speed of 20 rpm was used in the rotarod experiment. Mice were given a 7 to 10 minute rest interval to prevent stress and fatigue.

Male C57BL/6 mice were treated with diapocynin by oral gavage daily and then inflammatory, neurochemical, behavioral and histological studies were performed at various time intervals, as depicted in Figure  1A. To assess the neuroprotective effect of diapocynin, we first used two lower doses of diapocynin (100 and 150 mg/kg/day) and measured the dopamine level from striatum (Figure  1B). Both 100 and 150 mg/kg/day doses of diapocynin afforded some protection against MPTP-induced striatal dopamine loss, but it was not significant (Figure  1B). Therefore, we increased the dosage of diapocynin to 300 mg/kg/day and then evaluated the neuroprotective effects. Before determining the neuroprotective properties of diapocynin, the ability of diapocynin to cross the blood-brain barrier was examined. The LC/MS results showed a significant accumulation of diapocynin in the SN and striatum of the brain following the oral gavage treatment of diapocynin (300 mg/kg/day) (Figure  1C,D).

Following 300 mg/kg/day diapocynin treatment, mice were sacrificed and brains were processed for HPLC neurochemical analysis (Figure  1E,G). We quantified levels of dopamine and its metabolites, DOPAC and HVA, in striatum following diapocynin treatment. As shown in Figure  1E, MPTP injection led to a >75% reduction in striatal dopamine levels compared with the striata of control mice. Remarkably, diapocynin treatment significantly protected against MPTP-induced striatal dopamine depletion (Figure  1E). Diapocynin treatment also significantly restored DOPAC and HVA levels in MPTP-treated mice (Figure  1F,G).

In order to determine whether diapocynin interferes with the toxic metabolic conversion of MPTP to MPP⁺ by MAO-B, we measured the level of MPP⁺ in striatum 3 h after the final MPTP injection. We found that diapocynin had no effect on striatal levels of MPP⁺ (MPTP mice, 1860 ± 569 ng/g; MPTP plus diapocynin mice, 1775 ± 586 ng/g). Next, we also tested whether diapocynin treatment alone alters neurochemical levels in the striatum. As shown in Figure  1E,G, oral administration of diapocynin (300 mg/kg/day) alone for 12 days did not alter striatal dopamine levels. Also, diapocynin treatment did not produce behavioral abnormalities (data not shown).

Next, we determined if diapocynin protected against MPTP-induced microglial activation and astrogliosis. As shown in Figure  2, a significant increase in the expression of ionized calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) in SN of the MPTP-treated group was observed. DAB immunostaining of Iba-1 in MPTP nigral brain tissue showed an increased number of amoeboid-shaped microglial cells with thick processes, indicative of active microgliosis (Figure  2A). DAB immunostaining of GFAP in MPTP-treated mice showed excessive astrogliosis, as measured by increased size and processes thickness in GFAP-positive cells (Figure  2D).

Importantly, diapocynin strongly inhibited MPTP-induced microgliosis, as demonstrated by the very few Iba-1-positive microglial cells in the drug-treated group (Figure  2A). Also, diapocynin significantly decreased MPTP-induced increases in GFAP-positive astroglial cells in the mouse SN (Figure  2D). Western blot analysis for Iba-1 and GFAP also revealed that diapocynin suppresses nigral Iba-1 (Figure  2B and C) and GFAP (Figure  2E,F) protein expression in MPTP-treated mice. Together, these data suggest that diapocynin significantly attenuates glial cell activation in the nigral regions of the MPTP mouse model of PD.

iNOS, a key proinflammatory enzyme, is typically elevated in disease conditions [3]. We observed a marked increase in the expression of iNOS in the SN of MPTP-treated mice as compared to control mice (Figure  3A,B). However, diapocynin attenuated MPTP-induced expression of iNOS protein (Figure  3A,B). Additionally, immunofluorescence analysis for iNOS in the SN sections shows that MPTP treatment led to a marked increase in nigral iNOS protein expression, and that iNOS co-localized with GFAP-positive astrocytes and Iba-1-positive microglial cells (Figure  3C,D). Consistent with its inhibitory effect on glial cell activation, diapocynin suppressed MPTP-induced expression of iNOS (Figure  3C,D). These results demonstrate that diapocynin effectively suppresses the expression of the proinflammatory molecule iNOS in vivo in the SN of MPTP-treated mice.

NADPH oxidase is a major source of ROS in the brain. Recent findings suggest that NADPH oxidase-induced oxidative stress plays a central role in nigral dopaminergic neurodegeneration in PD patients and animal models [20]. Western blot analysis showed an increased expression of gp91phox, a key subunit of NADPH oxidase, after MPTP injection compared to weak expression in saline-treated mice (Figure  4A,B). Robust gp91phox immunoreactivity was seen specifically in larger cells with thick, shorter ramifications in the SN of MPTP-treated mice (Figure  4C). Double immunolabeling studies confirmed that gp91phox immunoreactivity appeared to co-localize with Iba-1-positive microglia (Figure  4C, middle panel). Diapocynin treatment attenuated MPTP-induced gp91phox protein expression in the Iba-1-positive microglial cells in the SN (Figure  4C). In the MPTP model of PD, gp91phox does not co-localize with either astrocytes or dopaminergic neurons [20]. These results suggest that diapocynin effectively blocks NADPH oxidase expression in response to MPTP.

3-Nitrotyrosine (3-NT) has been widely used as a marker of nitric oxide-dependent oxidative stress [21]. Western blot analysis demonstrated increased expression of 3-NT modified proteins in the SN of MPTP-treated mice, while diapocynin treatment significantly suppressed MPTP-induced 3-NT levels (Figure  5A,B). Further confirmation came from immunolabeling of 3-NT in the SN sections. In sections from MPTP-treated mice, a dramatic increase in the expression of 3-NT, specifically in the SN region of the ventral midbrain, was observed. Notably, 3-NT co-localized (yellow color) with TH-positive dopaminergic neurons (Figure  5C). However, 3-NT expression was observed in very few TH-positive dopaminergic neurons in the MPTP and diapocynin co-treated animals (Figure  5C). These results strongly suggest that diapocynin inhibits nitration of TH-positive dopaminergic neurons in the nigra during neurotoxic insult.

In addition to protein nitration, MPTP treatment significantly increased oxidative damage in the nigral regions, as measured by 4-HNE Western blot analysis (molecular weight of 68kDa) (Figure  5D,E). However, diapocynin strongly inhibited MPTP-induced expression of this unsaturated aldehyde in the SN (Figure  5D,E). Consistent with these findings, immunofluorescence analysis of the MPTP-treated SN sections showed a dramatic increase in 4-HNE formation in the dopaminergic neurons, as evidenced by TH and 4-HNE double immunolabeling (Figure  5F). Interestingly, diapocynin treatment abolished MPTP-induced 4-HNE generation in dopaminergic neurons (Figure  5F). These results suggest that diapocynin mitigates oxidative damage in nigral dopaminergic neurons following MPTP neurotoxicity.

Next, we determined whether diapocynin protects the dopaminergic neurons against MPTP toxicity. MPTP treatment led to degeneration of TH-positive dopaminergic neurons and terminals in the SN and striatum (Figure  6A, 2× magnification). Higher magnified 10× pictures (Additional file 1: Figure S1A) clearly demonstrated loss of neurons in substantia nigra pars compacta (SNpc), substantia nigra lateralis (SNl) and substantia nigra reticularis (SNr) regions of the nigral tract. Additionally, stereological counting of TH-positive neurons in SN of MPTP-treated mice also showed > 60% reduction (Figure  6E).

Interestingly, diapocynin treatment improved MPTP-induced damage of nigral TH-positive neurons and striatal TH terminals (Figure  6A,E and Additional file 1: Figure S1A). Consistent with these findings, Western blot of TH in nigra and striatum also showed significantly decreased TH protein levels in MPTP-treated mice (Figure  6B,C,D). However, orally administered diapocynin significantly prevented loss of nigral and striatal TH in MPTP-treated mice (Figure  6B,C,D). Further assessment of degenerating neurons in the nigral regions by FJB staining revealed that diapocynin-treated MPTP mice had fewer FJB-positive cells than untreated MPTP mice (Additional file 1: Figure S1B). Collectively, these results suggest that diapocynin is neuroprotective in the MPTP mouse model of PD.

To assess the effectiveness of diapocynin against motor deficits induced by MPTP, we measured various motor performance parameters using VersaMax infrared computerized activity monitoring system and a rotarod instrument (Accuscan). Behavioral function was assessed 4 days after the last dose of MPTP treatment. Representative motor activity maps of movement of saline-treated control, MPTP and MPTP plus diapocynin-treated mice are shown in Figure  7A. As expected, MPTP drastically decreased movement in all directions. Diapocynin treatment dramatically improved locomotion in the MPTP plus diapocynin-treated group. Further analysis of locomotor activity data revealed that subacute MPTP treatment markedly decreased horizontal activity (Figure  7B), vertical activity (Figure  7C), total distance travelled (Figure  7D), movement time (Figure  7E), observed stereotypies (Figure  7G), observed rearings (Figure  7H) and rotarod performances at 20 rpm speed (Figure  7I), consistent with our previous observations [16]. Additionally, rest time was increased in the MPTP-treated mice (Figure  7F). Notably, diapocynin treatment significantly restored MPTP-induced locomotor and motor co-ordination impairments for every endpoint measured (Figure  7B,C,D,E,F,G,H,I).

Following characterization of the neuroprotective effect of diapocynin in the typical subacute MPTP model, we further examined whether diapocynin can intervene the on-going degenerative processes in a post-treatment paradigm. In order to test the efficacy of diapocynin post-treatment, mice were treated with MPTP at a dose of 25 mg/kg/day for 3 days followed by co-treatment with MPTP and diapocynin (300 mg/kg/day) for 2 days. Mice also received another 6 doses of diapocynin (300 mg/kg/day) and were sacrificed for neurotransmitter analysis. As evident from Figure  8A,B,C, we observed significantly reduced striatal dopamine (75%), DOPAC (73%) and HVA (70%) in MPTP-treated mice. Importantly, diapocynin post-treatment showed a reduction of 52% of dopamine (Figure  8A), 50% of DOPAC (Figure  8B) and 40% of HVA (Figure  8C). Thus, these results suggest that diapocynin slows the progression of dopaminergic neurodegeneration in a post-treatment MPTP mouse model of PD.

Although the subacute MPTP model is very efficient for drug screening and elucidating molecular mechanisms, it does not recapitulate the chronic progression of degenerative processes associated with PD. In our experiment, we measured whether diapocynin efficiently protected nigrostriatum in a chronic MPTP model (Figure  8D,E,F). As expected, chronic MPTP administration led to approximately 80% loss of dopamine (Figure  8D), 60% loss of DOPAC (Figure  8E) and 70% loss of HVA (Figure  8F). Consistent with observations in the subacute MPTP model, diapocynin significantly restored dopamine (P <0.01; Figure  8D), DOPAC (P <0.05; Figure  8E) and HVA (P <0.05; Figure  8F) in striatum, demonstrating that oral diapocynin treatment can slow the progressive neurodegenerative process in the nigrostriatal dopaminergic system.