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, 36 (23), 6332-51

Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection Against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease

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Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection Against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease

Manuj Ahuja et al. J Neurosci.

Abstract

A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic.

Significance statement: Almost two centuries since its first description by James Parkinson, Parkinson's disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.

Keywords: MPTP; Nrf2; fumarates; inflammation; mitochondria; oxidative stress.

Figures

Figure 1.
Figure 1.
DMF forms an adduct with GSH and depletes intracellular GSH levels and decreases cell viability in a dose-dependent manner. Time course of the S-alkylation reaction between 1 mm DMF or 1 mm MMF with 1 mm GSH in PBS at pH 7.4 was measured as described in Materials and Methods. Time course for GSH consumption (A) and GS-DMF and GS-MMF adduct formation (B). C, Intracellular GSH, GSSG, and their ratio (GSH/GSSG) were determined at 24 h after DMF and MMF (10, 50, and 200 μm) treatment. Bars represent mean ± SEM. *p < 0.05 compared with controls; #p < 0.05 compared with DMF at 10 μm (n = 4). D, Cell viability in N27 rat dopaminergic cells treated with DMF (1, 10, 50, and 100 μm) or MMF (1, 10, 50, and 100 μm) for 24 h was assessed using Presto Blue cell viability kits. Bar plot represents the percentage of the control as mean ± SEM of viable cells (n = 3). *p < 0.05 compared with control (DMSO); #p < 0.05 compared with the DMF (1 μm)-treated group; $p < 0.05 compared with the DMF (50 μm)-treated group (n = 3).
Figure 2.
Figure 2.
DMF directly activates the Nrf2 pathway via S-alkylation. A, Dose-dependent activation of the Neh2–luc reporter by fumarate compounds (DMF and MMF) compared with the known Nrf2 activator TBHQ. B, Selectivity of fumarate compounds for Neh2–luc activation is shown compared with their activity toward the ODD–luc reporter versus the potent HIF activator ciclopirox. Experiments were performed in triplicate, and the mean ± SEM is plotted on the scatter plot. C, Quenching of the DMF dose–response curve in the Neh2–luc reporter assay during simultaneous incubation with thiol reagents: 0.5 mm N-acetyl cysteine (NAC), cysteine, or GSH. Each experiment was performed in triplicate. The plot represents values of mean ± SEM.
Figure 3.
Figure 3.
Nrf2 activation of DMF and MMF involves Bach1 nuclear export and is associated with Hmox1-mediated neuroprotection. A, Immunoblot showing total, nuclear, and cytosolic Bach1 and Nrf2 levels after a 2 and 4 h incubation with DMF (10 μm) or MMF (10 μm) in human BE(2)-M17 neuroblastoma cells. GAPDH was used as a loading control for the total fraction, whereas aldolase and PARP1 were used to verify the purity and to demonstrate equal loading of the cytosolic and nuclear fraction, respectively. B, Cell viability in N27 rat dopaminergic cells treated with DMF (10 μm) or MMF (10 μm) in the presence or absence of MPP+ and/or ZnPP. Plot represents percentage control as mean ± SEM of viable cells (n = 3). *p < 0.05 compared with DMSO control; #p < 0.05 compared with the MPP+-treated group; $p < 0.05 compared with the DMSO + ZnPP + MPP+-treated group; @p < 0.05 compared with the MPP+ + MMF-treated group.
Figure 4.
Figure 4.
DMF and MMF activate Nrf2/ARE signaling in vitro. A, B, qRT-PCR analysis showing relative mRNA levels of ARE-containing genes after DMF (20 μm; A) and MMF (20 μm; B) administration. Bars represent mean ± SEM. *p < 0.05 compared with respective controls (n = 3 per time point). Immunoblot analysis (C, D) and densitometry analysis (E, F) of ARE proteins and the changes in ARE-containing proteins after DMF (20 μm; C, E) and MMF (20 μm; D, F) treatment. Bars represent percentage control values depicted as mean ± SEM. *p < 0.05 compared with respective controls (n = 5 per time point).
Figure 5.
Figure 5.
Bioavailability of DMF and MMF and in vivo activation of ARE-containing genes by DMF. A, Levels of MMF in the brain, liver, and blood of C57BL/6 mice after two doses of 50 mg/kg (or 100 mg · kg−1 · d−1) DMF or MMF administered 12 h apart by oral gavage and measured at 6 h after the last dose. Bars represent mean ± SEM (n = 5 mice per group). *p < 0.05 compared with respective vehicle controls. B, Quantitative RT-PCR showing relative mRNA levels of ARE-containing genes in the ventral midbrain and liver after two doses of 50 mg/kg DMF administered 12 h apart by oral gavage and measured at 6 h after the last dose. Bars represent mean ± SEM (n = 5 mice per group). *p < 0.05 compared with vehicle controls.
Figure 6.
Figure 6.
Neuroprotective effects of DMF and MMF in the MPTP model of PD. Immunohistochemical staining for TH (A) and stereological analysis of total (Nissl) and TH+ neurons (B) in the SNpc in the acute MPTP model on day 7 after treatment with different doses of DMF (10, 50, and 100 mg · kg−1 · d−1, administered in 2 divided doses via oral gavage) for 6 d. Bars represent mean ± SEM. *p < 0.05 compared with saline controls; #p < 0.05 compared with MPTP (n = 6–10 mice per group). Striatal levels of DA and its metabolites DOPAC and HVA after DMF (C) or MMF (D) treatment as measured by HPLC-electrochemical detection analysis in the acute MPTP model on day 7. Bars represent mean ± SEM. *p < 0.05 compared with saline controls; #p < 0.05 compared with MPTP (n = 6–10 mice per group). Scale bar, 100 μm.
Figure 7.
Figure 7.
DMF attenuates MPTP-induced accumulation of oxidative stress and inflammation. A, Accumulation of oxidative stress in the substantia nigra demonstrated by 3-NT immunoreactivity 48 h after acute MPTP with and without DMF (100 mg · kg−1 · d−1) administration; representative images from n = 3 mice in each group. Scale bar, 100 μm. B, Quantitative comparison of area of 3-NT immunoreactivity in SNpc 48 h after MPTP and after DMF treatment. Bars represent mean ± SEM. *p < 0.05 compared with saline controls; #p < 0.05 compared with MPTP (n = 5). C, CD68-immunoreactive microglia in the substantia nigra 36 h after acute MPTP and treatment with DMF. Representative images from n = 3 mice in each group. Scale bar, 100 μm. D, CD68-positive microglial cell counts in the substantia nigra 36 h after acute MPTP and treatment with DMF. Bars represent mean ± SEM. *p < 0.05 compared with saline controls; #p < 0.05 compared with MPTP (n = 5). E, Levels of proinflammatory genes measured 24 h after the last dose of acute MPTP and after DMF treatment. Bars represent mean ± SEM. *p < 0.05 compared with saline controls; #p < 0.05 compared with MPTP (n = 5).
Figure 8.
Figure 8.
Nrf2-dependent neuroprotective effects of DMF and MMF against MPTP neurotoxicity. A, Immunohistochemical staining for TH in the SNpc of wild-type and Nrf2-KO mice on day 7 after DMF or MMF (50 mg/kg twice a day for 7 d via oral gavage) treatment in the acute MPTP model. Scale bar, 100 μm. B, Stereological analysis of total (Nissl) and TH+ neurons in the SNpc. Bars represent mean ± SEM. *p < 0.05 compared with wild-type controls; #p < 0.05 compared with wild-type MPTP; $p < 0.05 compared with KO control (n = 6 mice per group). C, Striatal levels of DA and its metabolites (HVA and DOPAC) measured by HPLC analysis in wild-type and Nrf2-KO mice. Bars represent mean ± SEM. *p < 0.05 compared with saline controls; #p < 0.05 compared with wild-type MPTP (n = 6 mice per group); $p < 0.05 compared with KO control.
Figure 9.
Figure 9.
Selective activation of the Nrf2 pathway by DMF. A, qRT-PCR analysis showing relative mRNA levels of ARE-containing genes in the liver and ventral midbrain of wild-type and Nrf2-KO mice 6 h after two doses of 50 mg/kg DMF administered by oral gavage 12 h apart. Bars represent fold expression of mRNA relative to β-actin values depicted as mean ± SEM. *p < 0.05 compared with respective controls of different ARE-containing genes; #p < 0.05 compared with the respective wild-type DMF treatment group (n = 5 mice per group). B, qRT-PCR analysis showing relative mRNA levels of ARE-containing genes in the wild-type and Nrf2-KO MEFs after treatment with DMF (20 μm) at different time points. Bars represent the mean ± SEM of relative mRNA levels (relative to β-actin). *p < 0.05 compared with 0 h respective controls (n = 3 per time point).
Figure 10.
Figure 10.
DMF and MMF modulate mitochondrial OCR in an Nrf2-dependent manner. A, Mitochondrial OCR in wild-type MEFs after incubation with DMF or MMF at 20 μm for 4 h (C) or 24 h. B, Mitochondrial OCR in Nrf2-KO MEFs after incubation with DMF or MMF at 20 μm for 4 h or 24 h (D). Bars represent the mean ± SEM of the OCR expressed in picomoles per minute per 2000 cells. *p < 0.05 compared with vehicle controls. Each experiment was performed three times and consisted of six samples per group. Basal Resp, Basal respiration; ATP synth, ATP synthesis; Max Resp, maximum respiration; Spare Cap, spare capacity; Non-Mito. Resp, non-mitochondrial respiration.
Figure 11.
Figure 11.
Modulation of ECAR by DMF and MMF in an Nrf2-dependent manner. A, ECAR in wild-type MEFs after incubation with DMF or MMF at 20 μm for 4 h or 24 h (C). B, ECAR in Nrf2-KO MEFs after incubation with DMF or MMF at 20 μm for 4 h or 24 h (D). Bars represent the mean ± SEM ECAR expressed in mpH per minute. *p < 0.05 compared with vehicle controls (n = 6). Each experiment was performed three times and consisted of six samples per group.
Figure 12.
Figure 12.
DMF and MMF increase mtDNA copy number and OXPHOS content in an Nrf2-dependent manner. A, Relative levels of the mtDNA variant of cytochrome c oxidase 1 gene were assessed in DMF-treated (20 μm) and MMF-treated (20 μm) wild-type and Nrf2-KO MEFs to determine the relative mitochondrial copy number. Cells were treated for 24 h, and mRNA levels were measured by qRT-PCR analysis. Bars represent the mean ± SEM of mRNA levels (relative to β-actin). *p < 0.05 compared with wild-type vehicle control; #p < 0.05 compared with Nrf2-KO vehicle control (n = 3). Immunoblot analysis (B) and densitometry analysis (C) of different subunits of complexes involved in mitochondrial electron transport chain after DMF (20 μm) or MMF (20 μm) treatment for 24 h in MEFs. Bars represent percentage control values depicted as mean ± SEM. *p < 0.05 compared with wild-type vehicle control; #p < 0.05 compared with respective Nrf2-KO vehicle control (n = 5). mitochondrial COX-I, Mitochondrial cytochrome c oxidase I.
Figure 13.
Figure 13.
DMF and MMF induce mitochondrial biogenesis in an Nrf2-dependent manner. A, B, Relative mRNA levels of mitochondrial biogenesis genes in DMF-treated (20 μm) or MMF-treated (20 μm) wild-type and Nrf2-KO MEFs. Cells were treated for 24 h, and mRNA levels were measured by qRT-PCR analysis. Bars represent the mean ± SEM of mRNA levels (relative to β-actin). *p < 0.05 compared with wild-type vehicle control; #p < 0.05 compared with respective KO vehicle control (n = 3). mt-Nd1, mitochondrial-encoded NADH dehydrogenase 1; mt-Nd2, mitochondrial-encoded NADH dehydrogenase 2; mt-Nd5, mitochondrial-encoded NADH:ubiquinone oxidoreductase core subunit 5; mt-Nd6, mitochondrial-encoded NADH:ubiquinone oxidoreductase core subunit 6; mt-Cytb, mitochondrial cytochrome b; mtCo3, mitochondrial cytochrome c oxidase subunit III; Ssbp1, single-stranded DNA binding protein 1, mitochondrial; Mterf1a, mitochondrial transcription termination factor 1a; Mterf3, mitochondrial transcription termination factor 3; Tfb1m, transcription factor B1, mitochondrial; Tfb2m, transcription factor B2, mitochondrial; Tfam, transcription factor A, mitochondrial; Polg2, polymerase (DNA directed), γ2, accessory subunit; Polrmt, polymerase (RNA) mitochondrial (DNA directed); Peo1, progressive external ophthalmoplegia 1 (Twinkle protein, mitochondrial).
Figure 14.
Figure 14.
Mechanism of Nrf2 activation by DMF and MMF and its role in neuroprotection. DMF and MMF differentially activate the Nrf2/ARE pathway to modulate the antioxidant, anti-inflammatory, and mitochondrial biosynthetic machinery. 1, Under basal conditions, Nrf2 is sequestered in the cytoplasm through its binding to its cytoplasmic inhibitor Keap1, which promotes Nrf2 degradation via a ubiquitin proteasome pathway. 2, In the presence of electrophilic agents such as DMF and MMF, the cysteine residues on Keap1 are modified as a result of an alkylation or reduction process, which in turns prevents the ubiquitin-dependent proteasomal degradation of Nrf2. Both DMF and MMF in our study have been shown to possess strong alkylation properties toward thiol groups present on GSH; this characteristic might explain their ability to disrupt the Keap1–Nrf2 interaction by alkylating the cysteine residues on Keap1, leading to nuclear translocation of Nrf2. 3, 4, The Nrf2 pathway is kept in check normally by Bach1, which interacts with small Maf proteins in the nucleus and acts as a repressor of Nrf2-induced ARE-containing gene activation. Once inside the nucleus, Nrf2 binds to the ARE sites of the ARE-containing genes after the export of Bach1 from the nucleus to the cytoplasm. 5, After the export of Bach1 from the nucleus, Nrf2 complexes with small Maf proteins, induces ARE activation, and upregulates a battery of genes involved in antioxidant and anti-inflammatory responses. 6, Both DMF and MMF demonstrate activation of the Nrf2 pathway, although differentially. 7, In our study, we also found that DMF and MMF mediated mitochondrial biogenesis in an Nrf2-dependent manner via an unknown mechanism, accompanied by enhanced cellular bioenergetics to render neuroprotection. Nrf2 activation by DMF, but not MMF, was associated with depletion of GSH, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro.

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