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. 2011 Jul 1;51(1):88-96.
doi: 10.1016/j.freeradbiomed.2011.03.027. Epub 2011 Mar 30.

Neuroprotective effect of Nrf2/ARE activators, CDDO ethylamide and CDDO trifluoroethylamide, in a mouse model of amyotrophic lateral sclerosis

Arie Neymotin  1 , Noel Y Calingasan, Elizabeth Wille, Nima Naseri, Susanne Petri, Maria Damiano, Karen T Liby, Renee Risingsong, Michael Sporn, M Flint Beal, Mahmoud Kiaei
Affiliations

Neuroprotective effect of Nrf2/ARE activators, CDDO ethylamide and CDDO trifluoroethylamide, in a mouse model of amyotrophic lateral sclerosis

Arie Neymotin et al. Free Radic Biol Med. .

Abstract

Oxidative damage, neuroinflammation, and mitochondrial dysfunction contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS), and these pathologic processes are tightly regulated by the Nrf2/ARE (NF-E2-related factor 2/antioxidant response element) signaling program. Therefore, modulation of the Nrf2/ARE pathway is an attractive therapeutic target for neurodegenerative diseases such as ALS. We examined two triterpenoids, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) ethylamide and CDDO trifluoroethylamide (CDDO-TFEA), that potently activate Nrf2/ARE in a cell culture model of ALS and in the G93A SOD1 mouse model of ALS. Treatment of NSC-34 cells stably expressing mutant G93A SOD1 with CDDO-TFEA upregulated Nrf2 expression and resulted in translocation of Nrf2 into the nucleus. Western blot analysis showed an increase in the expression of Nrf2/ARE-regulated proteins. When treatment started at a "presymptomatic age" of 30days, both of these compounds significantly attenuated weight loss, enhanced motor performance, and extended the survival of G93A SOD1 mice. Treatment started at a "symptomatic age," as assessed by impaired motor performance, was neuroprotective and slowed disease progression. These findings provide further evidence that compounds that activate the Nrf2/ARE signaling pathway may be useful in the treatment of ALS.

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Figures

Figure 1
Figure 1. Chemical structures of amide derivatives of CDDO
Figure 2
Figure 2
A) CDDO-TFEA treatment increased Nrf2 expression in NSC-34 cells. NSC-34 cells with and without the G93A SOD1 mutation were seeded on glass-bottom chamber slides and treated with CDDO-TFEA (300 nmolar) for 24 hours. Cells were fixed and stained for Nrf2 using anti-Nrf2 (Abcam). This data is representative of three experiments. B) Nrf2/ARE gene upregulation in NSC-34 G93A SOD1 cells treated with CDDO-TFEA. NSC-34 cells were grown on plastic tissue culture plates and allowed to grow to confluency. These cells were treated with CDDO-TFEA or DMSO (vehicle) for 24 hours. Cells were washed and total RNA was extracted using TRIZOL. RNAs were analyzed by real time quantitative RT-PCR using TaqMan assays and Applied Biosystem PCR systems. Nrf2-regulated genes like NQO1, GST-a3 and HO-1 were significantly upregulated (p<0.0001, student t-test). Beta actin TaqMan assay was used as house-keeping gene to compare and calculate the fold difference in control and treated cells. All experiments were done in triplicates.
Figure 2
Figure 2
A) CDDO-TFEA treatment increased Nrf2 expression in NSC-34 cells. NSC-34 cells with and without the G93A SOD1 mutation were seeded on glass-bottom chamber slides and treated with CDDO-TFEA (300 nmolar) for 24 hours. Cells were fixed and stained for Nrf2 using anti-Nrf2 (Abcam). This data is representative of three experiments. B) Nrf2/ARE gene upregulation in NSC-34 G93A SOD1 cells treated with CDDO-TFEA. NSC-34 cells were grown on plastic tissue culture plates and allowed to grow to confluency. These cells were treated with CDDO-TFEA or DMSO (vehicle) for 24 hours. Cells were washed and total RNA was extracted using TRIZOL. RNAs were analyzed by real time quantitative RT-PCR using TaqMan assays and Applied Biosystem PCR systems. Nrf2-regulated genes like NQO1, GST-a3 and HO-1 were significantly upregulated (p<0.0001, student t-test). Beta actin TaqMan assay was used as house-keeping gene to compare and calculate the fold difference in control and treated cells. All experiments were done in triplicates.
Figure 3
Figure 3. Western blot analysis of NSC-34/G93A A) Cell lysates for Nrf2/ARE proteins. B) Cytoplasmic and nuclear fractions for Nrf2 protein
NSC-34/G93A cells were plated and treated with CDDO-TFEA or DMSO as described in Figure 2. Cell lysates were prepared at the indicated time points. A) Lanes 1, 3, and 5 each contain 30 µg of total lysates from CDDO-TFEA-treated cells and lanes 2, 4, and 6 each contain 30 µg of total lysates from DMSO-treated cells. Nrf2 is upregulated as early as 8 hours. NQO1, HO1 and GR were upregulated and reached peak levels at 48 hours. Beta-actin, a house-keeping protein is shown for a loading control. B) Lanes 1 and 3 are cytoplasmic (C) and lanes 2 and 4 are nuclear (N) fractions, 20 µg per lane loaded. . SOD1 used as cytoplasmic protein.
Figure 3
Figure 3. Western blot analysis of NSC-34/G93A A) Cell lysates for Nrf2/ARE proteins. B) Cytoplasmic and nuclear fractions for Nrf2 protein
NSC-34/G93A cells were plated and treated with CDDO-TFEA or DMSO as described in Figure 2. Cell lysates were prepared at the indicated time points. A) Lanes 1, 3, and 5 each contain 30 µg of total lysates from CDDO-TFEA-treated cells and lanes 2, 4, and 6 each contain 30 µg of total lysates from DMSO-treated cells. Nrf2 is upregulated as early as 8 hours. NQO1, HO1 and GR were upregulated and reached peak levels at 48 hours. Beta-actin, a house-keeping protein is shown for a loading control. B) Lanes 1 and 3 are cytoplasmic (C) and lanes 2 and 4 are nuclear (N) fractions, 20 µg per lane loaded. . SOD1 used as cytoplasmic protein.
Figure 4
Figure 4. Nrf2 upregulation and translocation in rat embryonic motor neurons in response to CDDO-TFEA
Rat embryonic motor neurons (Rat eMNs) were isolated as described in the Methods section. Rat eMNs were plated and treated with 300 nM CDDO-TFEA (Figure 4B, D) or DMSO (Figure 4A, C) for 24 hours. These cells were washed, fixed and then stained for Nrf2 using anti-Nrf2 (Figure 4A,B). Double immunofluorescence was performed to study nuclear translocation of Nrf2. CDDO-TFEA incubation led to a reduction of cytoplasmatic Nrf2 staining (green fluorescence) and increased nuclear colocalization (Figure 4 C,D).
Figure 5
Figure 5. CDDOs specifically activate the Nrf2/ARE signaling program
Wild-type and Nrf2 knockout mouse embryonic fibroblasts were pre-treated with either CDDO-EA or CDDO-TFEA (1, 10, 100 and 300 nM) or DMSO (as control) overnight. Then all of these cells were treated with 250 µM of tert-butylhydroperoxide (tBHP). Generation of reactive oxygen species (ROS) was measured 15 minutes later by flow cytometry. CDDO-EA or CDDO-TFEA treatments resulted in a significant dose-dependent reduction of tert-butylhydroperoxide-induced ROS generation in wild-type fibroblasts. Nrf2 knockout fibroblasts failed to show a reduction in tBHP-induced ROS generation. This data is representative of three separate experiments. *** indicates a significant increase in ROS production caused by tBHP treatment compared to DMSO treatment (p<0.0001). ## indicates significant reduction in ROS in CDDO-EA or CDDO TFEA pre-treated as wild-type cells compared to Nrf2 KO cells (p<.001, ANOVA).
Figure 6
Figure 6. Brain levels of triterpenoids measured by LC-MS
Twelve week old male C57Bl6 mice were fed by gavage once with 100 microlitters of 2 micromolar CDDO-EA or CDDO TFEA dissolved in Neobee oil. The CDDOs were measured in the brain after 6, 24 and 48 hours of gavage. Data represent Mean ± SEM, (n=5 mice in each group for three time points). Animals fed with vehicle showed no triterpenoids in the brain (data not shown).
Figure 7
Figure 7. CDDO-EA and CDDO-TFEA treatment cause translocation of Nrf2 from the cytoplasm to the nucleus in motor neurons in the spinal cords of ALS mice
G93A SOD1 mice were fed food containing CDDO-EA or CDDO-TFEA (400 mg/kg) for 10 days. Non-transgenic controls and G93A controls were fed control food. Spinal cord sections were prepared and stained with anti-Nrf2. Non-transgenic control spinal cord sections were weakly stained with anti-Nrf2 in the cytoplasm. G93A control spinal cord sections show mild Nrf2 staining. Larger motor neurons show Nrf2 only in the cytoplasm, while some smaller neurons/glial cells show nuclear staining. G93A mice treated with CDDO-EA or CDDO-TFEA produced strong nuclear staining for Nrf2 (n=5 per group). Arrows point to the nucleus (lightly stained in controls and darkly stained in CDDO-EA or CDDO-TFEA panels).
Figure 8
Figure 8
A: Nrf2/ARE activation is upregulated by antioxidant genes in G93A spinal cord following triterpenoid treatment. Seventy-five day old G93A SOD1 transgenic mice were fed with either vehicle or CDDO-EA or CDDO-TFEA (400 mg/kg in food) daily for ten days. Spinal cords were analyzed for genes involved in antioxidant response by quantitative real time RT-PCR. Data represent Mean ± SEM (n=4, *p<0.001). Beta actin or GAPDH TaqMan assays were used as control genes in real time RT-PCR to compare the difference in expression level of antioxidant genes in samples from treated and untreated G93A SOD1 mice. Student’s t-test compared to control. B: Nrf2/ARE activation upregulated inflammatory genes in G93A spinal cord following triterpenoid treatment. Seventy-five day old G93A SOD1 transgenic mice were fed with either vehicle or CDDO-EA or CDDO-TFEA (400 mg/kg in food) daily for ten days. Spinal cords were analyzed for genes involved in inflammatory response by quantitative real time RT-PCR. Data represent Mean ± SEM (n=4, *p<0.001). Beta actin or GAPDH TaqMan assays were used as control genes in real time RT-PCR to compare the difference in expression level in inflammatory genes in samples from treated and untreated G93A SOD1 mice. Student’s t-test compared to control. C: Nrf2/ARE activation upregulated mitochondrial biogenesis genes in G93A spinal cord following triterpenoid treatment. Seventy-five day old G93A SOD1 transgenic mice were fed with either vehicle or CDDO-EA or CDDO-TFEA (400 mg/kg in food) daily for ten days. Spinal cords were analyzed for genes involved in mitochondrial biogenesis by quantitative real time RT-PCR. Data represent Mean ± SEM (n=4, *p<0.001). Beta actin or GAPDH TaqMan assays were used as control genes in real time RT-PCR to compare the difference in expression level in mitochondrial enhancing genes in samples from treated and untreated G93A SOD1 mice. Students t-test compared to control.
Figure 8
Figure 8
A: Nrf2/ARE activation is upregulated by antioxidant genes in G93A spinal cord following triterpenoid treatment. Seventy-five day old G93A SOD1 transgenic mice were fed with either vehicle or CDDO-EA or CDDO-TFEA (400 mg/kg in food) daily for ten days. Spinal cords were analyzed for genes involved in antioxidant response by quantitative real time RT-PCR. Data represent Mean ± SEM (n=4, *p<0.001). Beta actin or GAPDH TaqMan assays were used as control genes in real time RT-PCR to compare the difference in expression level of antioxidant genes in samples from treated and untreated G93A SOD1 mice. Student’s t-test compared to control. B: Nrf2/ARE activation upregulated inflammatory genes in G93A spinal cord following triterpenoid treatment. Seventy-five day old G93A SOD1 transgenic mice were fed with either vehicle or CDDO-EA or CDDO-TFEA (400 mg/kg in food) daily for ten days. Spinal cords were analyzed for genes involved in inflammatory response by quantitative real time RT-PCR. Data represent Mean ± SEM (n=4, *p<0.001). Beta actin or GAPDH TaqMan assays were used as control genes in real time RT-PCR to compare the difference in expression level in inflammatory genes in samples from treated and untreated G93A SOD1 mice. Student’s t-test compared to control. C: Nrf2/ARE activation upregulated mitochondrial biogenesis genes in G93A spinal cord following triterpenoid treatment. Seventy-five day old G93A SOD1 transgenic mice were fed with either vehicle or CDDO-EA or CDDO-TFEA (400 mg/kg in food) daily for ten days. Spinal cords were analyzed for genes involved in mitochondrial biogenesis by quantitative real time RT-PCR. Data represent Mean ± SEM (n=4, *p<0.001). Beta actin or GAPDH TaqMan assays were used as control genes in real time RT-PCR to compare the difference in expression level in mitochondrial enhancing genes in samples from treated and untreated G93A SOD1 mice. Students t-test compared to control.
Figure 9
Figure 9
A: CDDO-EA treatment extends survival in G93A SOD1 mice. G93A SOD1 mice (n=18) were treated with CDDO-EA (starting at 30 days of age). The control group (n=18) was treated with control food (Chaw diet # 5002). The data were analyzed and compared using the Kaplan-Meier plot. Survival is significantly longer in CDDO-EA treated G93A SOD1 mice vs controls (p=0.001, Logrank, Mental-Cox). B: CDDO-TFEA treatment extends survival in G93A SOD1 mice. G93A SOD1 mice (n=18) were treated with CDDO-TFEA (starting at 30 days of age). The control group (n=18) was treated with control food (Chaw diet # 5002). The data were analyzed and compared using the Kaplan-Meier plot. Survival is significantly longer in CDDO-TFEA-treated G93A SOD1 mice vs controls (P=0.001, Logrank, Mental-Cox).
Figure 10
Figure 10. A) Treatment with CDDO-EA and B) CDDO-TFEA from the onset of ALS extends survival in G93A SOD1 mice
G93A SOD1 mice (n=18) were treated with CDDO-EA or CDDO-TFEA from the age of onset of ALS. The control group (n=18) was treated with control food (Chaw diet # 5002). The data were analyzed and compared using the Kaplan-Meier plot. Survival is significantly longer for CDDO-EA and CDDO-TFEA-treated G93A SOD1 mice vs controls (P=0.001, Logrank, Mental-Cox).
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