Neuroprotective effects of the triterpenoid, CDDO methyl amide, a potent inducer of Nrf2-mediated transcription.

The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. We tested neuroprotective effects of the synthetic triterpenoid CDDO-MA, a potent activator of the Nrf2/ARE signaling. CDDO-MA treatment of neuroblastoma SH-SY5Y cells resulted in Nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of ARE pathway genes. CDDO-MA blocked t-butylhydroperoxide-induced production of reactive oxygen species (ROS) by activation of ARE genes only in wild type, but not Nrf2 knockout mouse embryonic fibroblasts. Oral administration of CDDO-MA resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological alpha-synuclein accumulation and oxidative damage in mice. Additionally, CDDO-MA treatment in rats produced significant rescue against striatal lesions caused by the neurotoxin 3-NP, and associated increases in the oxidative damage markers malondialdehyde, F(2)-Isoprostanes, 8-hydroxy-2-deoxyguanosine, 3-nitrotyrosine, and impaired glutathione homeostasis. Our results indicate that the CDDO-MA renders its neuroprotective effects through its potent activation of the Nrf2/ARE pathway, and suggest that triterpenoids may be beneficial for the treatment of neurodegenerative diseases like Parkinson's disease and Huntington's disease

CDDO-MA attenuates MPTP-induced nigrostriatal dopaminergic neurodegeneration.

<p>Mice were pre-treated with either CDDO-MA or control diet 7 days before acute (MPTP 10 mg/kg, i.p. 3 doses every 2 hours in one day) and chronic (MPTP 40 mg/kg, for 28 day through continuous subcutaneous infusion using mini-osmotic pumps) MPTP treatment. Control animals received identical volumes of PBS. <i>A</i> Photomicrographs of TH-immunostained sections through the SNpc of mice on acute MPTP show a significant reduction in TH-positive neurons of SNpc. CDDO-MA treatment significantly blocked MPTP-induced loss of TH-positive neurons. <i>B.</i> Consistent with TH-immunostaining of SNpc, stereologic cell counts of total (Nissl-positive) and TH-immunopositive neurons of SNpc showed a significant loss following MPTP administration which was significantly attenuated by CDDO-MA treatment. <i>C.</i> Photomicrographs of TH-immunostained sections through the SNpc of mice on chronic MPTP show a significant reduction in TH-positive neurons of SNpc. CDDO-MA treatment significantly blocked MPTP-induced loss of TH-positive neurons. <i>D.</i> Consistent with TH-immunostaining of SNpc, stereologic cell counts of total (Nissl-positive) and TH-immunopositive neurons of SNpc showed a significant loss following MPTP administration which was significantly attenuated by CDDO-MA treatment. n = 10 mice per group. ^#<i>p</i><0.05 compared to control diet alone; *<i>P</i><0.05 when compared with the control diets with MPTP group. <i>E.</i> Photomicrographs of malondialdehyde (MDA)-immunostained sections through the SNpc of mice on chronic MPTP show a significant increase of MDA staining in SNpc of MPTP treated animals. CDDO-MA treatment resulted in a marked reduction of MPTP-induced MDA formation. Representative image from n = 5 mice in each group. <i>F.</i> Photomicrographs of α-synuclein stained sections through the SNpc of mice on chronic MPTP show a significant increase of α-synuclein accumulation in SNpc of MPTP treated animals. CDDO-MA treatment resulted in a significant reduction of MPTP-induced α-synuclein accumulation in SNpc neurons. High magnification inserts show the α-synuclein staining in neurons. Representative image from n = 5 mice in each group.</p

CDDO-MA treatment to SH-SY5Y cells causes Nrf2 translocation to nucleus and subsequent activation of ARE pathway.

<p><i>A.</i> Chemical structures of oleanolic acid and CDDO-MA. Oleanolic acid is a naturally occurring triterpenoid with structural similarities to CDDO. CDDO that is over 200,000 times more potent than the parent oleanolic acid was chemically modified by addition of a methyl group to obtain increased brain bioavailability. <i>B.</i> SH-SY5Y neuroblastoma cells were treated with CDDO-MA (1 µM) or DMSO as a control. In DMSO controls Nrf2 is localized to cytosol as immunostained by anti-Nrf2 antibody (closed arrow) with DAPI (open arrow) a nuclear stain. After 8 hours of CDDO-MA treatment Nrf2 from cytosol translocates to the nucleus and colocalizes with DAPI (straight arrow), Scale bar 12 µm. <i>C.</i> Nuclear translocation of Nrf2 results in a significant increase in mRNA levels of Nrf2 and other ARE genes such as NQO-1, glutathione reductase (GR) and HO-1 measured by RT-PCR at 8 and 24 hours after drug treatment. Data represented as Mean±SEM showing relative levels of mRNA at various time points compared to DMSO controls, **p<0.05 and ***p<0.001. Students-<i>t</i> test, n = 5 samples for each experimental groups. <i>D and E.</i> Increases in mRNA levels of Nrf2 and ARE genes showed corresponding increases in levels of their respective proteins analyzed by western blotting and densitometry at 8 and 24 hours after CDDO-MA treatment. Data represented as Mean±SEM showing relative percent change in levels of protein normalized to actin at various time points compared to DMSO controls, **p<0.05 and ***p<0.001. Students-<i>t</i> test, n = 6 samples for each experimental groups. <i>F.</i> CDDO-MA treatment resulted in a significant dose-response increase in levels of reduced glutathione (GSH) measured at 24 hours. Data represented as Mean±SEM showing GSH levels at 24 hours compared to DMSO controls, **p<0.05 and ***p<0.001 following Students <i>t</i>-test (n = 6). Values represented as nanomoles/mg protein from total cell lysate.</p

Neuroprotective effects of CDDO-MA on striatal dopamine and its metabolites in MPTP neurotoxicity.

a<p>Data are expressed as mean±SEM. Values represent as ng per mg protein.</p>b<p>#, p<0.05, ##, p<0.01compared to PBS groups; *, p<0.05, **, p<0.01 compared to control diet +MPTP group, ANOVA, Student-Newman-Keul's post test.</p

Brain bioavailability of CDDO-MA in mice.

<p>CDDO-MA shows pharmacodynamically meaningful levels in brain. A dose-dependent increase in concentration of CDDO-MA was observed using an LC-MS analysis of acetonitrile brain extracts measured following administration CDDO-MA (at 200, 400 and 800 mg/kg of rodent diet) for one week, n = 5 mice per group. Vehicle diet consisting of 1∶3 (ethanol∶Neobee oil) showed no CDDO-MA levels in brain (data not shown).</p

Activation of Nrf2/ARE pathway by CDDO-MA is Nrf2 dependent.

<p><i>A.</i> Wild type (WT) and Nrf2 knockout (KO) mouse embryonic fibroblasts were pretreated with either CDDO-MA (1, 10 and 100 nM) or DMSO (as control) for 18 hours. Analysis of ARE genes showed a statistically significant increase in GSTa3, HO-1 and NQO-1 in wild type fibroblasts compared to DMSO treated controls. Nrf2 knockout fibroblasts failed to induce ARE genes following treatment with CDDO-MA compared to respective DMSO controls, implicating that CDDO-MA requires Nrf2 to activate ARE signaling. Data represent mean±SEM, *<i>P</i><0.001 when compared with the controls, by ANOVA, n = 3 from two separate experiments. <i>B.</i> Wild type and Nrf2 knockout mouse embryonic fibroblasts were pretreated with either CDDO-MA (1, 10 and 100 nM) or DMSO (as control) for 18 hours. The next day, 250 µM tert-butyl hydroperoxide was added to these fibroblasts for 15 minutes, and generation of ROS was measured by flow cytometry. CDDO-MA treatment resulted in a significant dose dependent reduction of tBHP-induced ROS generation in wild type fibroblasts. Nrf2 knockout fibroblasts failed to show reduction in tBHP-induced ROS generation. *p<0.05 compared to knockout fibroblasts; #p<0.05 compared to 1 nM CDDO-MA, by ANOVA, n = 3 from two separate experiments.</p

CDDO-MA reduces striatal damage caused by 3-NP toxicity, and blocks 3-NP induced striatal glutathione depletion.

<p>Lewis rats were pre-treated with either CDDO-MA diet (800 mg/kg) or control diet 7 days followed by continuous infusion of 3-NP (50 mg/kg/day) delivered through subcutaneously implanted miniosmotic pumps for 7 days. <i>A.</i> Photomicrographs of NeuN-immunostained sections through the coronal section of striatum and cortex of rats treated with control diet+PBS (left), control diet with 3-NP (middle) and CDDO-MA diet with 3-NP (right) show a significant loss of NeuN positive cells in 3-NP treated cortex and caudate compared to PBS controls. CDDO-MA treatment significantly reduced 3-NP induced loss of cortical and caudate NeuN positive cells. High magnifications in lower panel show the striatal neuronal loss caused by 3-NP and preservation by CDDO-MA. <i>B.</i> Consistent with loss of NeuN positive cells in cortex and the caudate, measurement of lesion volume in coronal brain slices of 3-NP treated controls show a significant increase in striatal lesion volumes. The administration of CDDO-MA significantly reduces striatal lesion volumes caused by 3-NP. Data represent mean±SEM. *<i>p</i><0.01, statistical significance compared to 3-NP treated controls, by student-<i>t</i> test, n = 15 rats per group. <i>C.</i> Rats described above were also used for analysis of striatal levels of reduced and oxidized glutathione. 3-NP administration resulted in a statistically significant reduction of striatal levels of reduced glutathione (GSH) and the ratio of reduced and oxidized glutathione (GSH/GSSG), whereas levels of oxidized glutathione (GSSG) were not significantly affected by 3-NP treatment measured by HPLC-electrochemistry. CDDO-MA treatment significantly blocked 3-NP induced reductions in striatal levels of GSH and the ratio of GSH/GSSG without impacting the GSSG levels. Data represent mean±SEM. *<i>p</i><0.05, statistical significance compared to controls and ^#<i>p</i><0.05 compared to 3-NP treatment, by ANOVA, n = 10 rats per group.</p

CDDO-MA treatment blocks 3-NP induced oxidative damage.

<p>Lewis rats described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005757#pone-0005757-g004" target="_blank">figure 4</a> were also used for striatal and cortical levels of oxidative damage markers. <i>A.</i> Photomicrographs of malondialdehyde (MDA)-immunostained coronal sections of the striatum of rats administered with 3-NP shows an increase in MDA staining in striatum. Treatment with CDDO-MA markedly reduced 3-NP induced MDA immunostaining in the striatum, n = 5 mice per group, scale bar: 100 µm <i>B.</i> Consistent with the striatal immunostaining of MDA, HPLC-electrochemical analysis of MDA levels in striatum and cortex show 3-NP resulted in a statistically significant increase in MDA levels. CDDO-MA treatment significantly reduced 3-NP induced MDA formation in striatum and cortex. Data represent mean±SEM. *<i>p</i><0.05 compared to normal controls and ^#<i>p</i><0.05 compared to 3-NP treatment, by ANOVA, n = 10 rats per group. <i>C.</i> Gas chromatography/Mass spectrometric analysis of esterified F₂-Isoprostanes (F₂-IsoPs) in cerebral cortex from rats treated with 3-NP showed significant increases in F₂-IsoPs levels. CDDO-MA treatment significantly abrogated 3-NP induced cortical increases in F₂-IsoPs levels. Data represent mean±SEM. *<i>p</i><0.05 compared to normal controls and ^#<i>p</i><0.05 compared to 3-NP treatment, by ANOVA, n = 10 rats per group. <i>D.</i> HPLC-electrochemical analysis of the ratio of 8OH2dG over dG shows a significant increase in oxidative DNA damage in the cerebral cortex following 3-NP treatment which is significantly attenuated due to CDDO-MA administration. Data represent mean±SEM. *<i>p</i><0.05 compared to normal controls and ^#<i>p</i><0.05 compared to 3-NP treatment, by ANOVA, n = 10 rats per group. <i>E.</i> Photomicrographs of 3-nitrotyrosine-immunostained coronal brain sections through the striatum of rats show a significant elevation in 3-nitrotyrosine levels (a marker of protein nitration) due to 3-NP administration. Treatment of CDDO-MA significantly reduced 3-NP induced increase in immunostaining of striatal 3-nitrotyrosine, n = 5 rats per group, scale bar: 100 µm.</p