Aberrant regulation of the GSK ‐3β/ NRF 2 axis unveils a novel therapy for adrenoleukodystrophy
2018; Springer Nature; Volume: 10; Issue: 8 Linguagem: Inglês
10.15252/emmm.201708604
ISSN1757-4684
AutoresPablo Ranea‐Robles, Nathalie Launay, Montserrat Ruíz, Noel Y. Calingasan, Magali Dumont, Alba Naudí, Manuel Portero‐Otín, Reinald Pamplona, Isidró Ferrer, M. Flint Beal, Stéphane Fourcade, Aurora Pujol,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoResearch Article11 July 2018Open Access Source DataTransparent process Aberrant regulation of the GSK-3β/NRF2 axis unveils a novel therapy for adrenoleukodystrophy Pablo Ranea-Robles Pablo Ranea-Robles orcid.org/0000-0001-6478-3815 Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Search for more papers by this author Nathalie Launay Nathalie Launay Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Search for more papers by this author Montserrat Ruiz Montserrat Ruiz Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Search for more papers by this author Noel Ylagan Calingasan Noel Ylagan Calingasan Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Magali Dumont Magali Dumont UMR S 1127, Inserm, U1127, CNRS, UMR 7225, Institut du Cerveau et de la Moelle épinière, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Alba Naudí Alba Naudí Experimental Medicine Department, University of Lleida-IRB Lleida, Lleida, Spain Search for more papers by this author Manuel Portero-Otín Manuel Portero-Otín Experimental Medicine Department, University of Lleida-IRB Lleida, Lleida, Spain Search for more papers by this author Reinald Pamplona Reinald Pamplona Experimental Medicine Department, University of Lleida-IRB Lleida, Lleida, Spain Search for more papers by this author Isidre Ferrer Isidre Ferrer Department of Pathology and Experimental Therapeutics, Faculty of Medicine, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain Center for Biomedical Research on Neurodegenerative Diseases (CIBERNED), ISCIII, Madrid, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain IDIBELL-Bellvitge University Hospital, L'Hospitalet de Llobregat, Spain Search for more papers by this author M Flint Beal M Flint Beal Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Stéphane Fourcade Corresponding Author Stéphane Fourcade [email protected] orcid.org/0000-0002-8031-5007 Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Search for more papers by this author Aurora Pujol Corresponding Author Aurora Pujol [email protected] orcid.org/0000-0002-9606-0600 Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain Search for more papers by this author Pablo Ranea-Robles Pablo Ranea-Robles orcid.org/0000-0001-6478-3815 Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Search for more papers by this author Nathalie Launay Nathalie Launay Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Search for more papers by this author Montserrat Ruiz Montserrat Ruiz Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Search for more papers by this author Noel Ylagan Calingasan Noel Ylagan Calingasan Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Magali Dumont Magali Dumont UMR S 1127, Inserm, U1127, CNRS, UMR 7225, Institut du Cerveau et de la Moelle épinière, Sorbonne Universités, UPMC Université Paris 06, Paris, France Search for more papers by this author Alba Naudí Alba Naudí Experimental Medicine Department, University of Lleida-IRB Lleida, Lleida, Spain Search for more papers by this author Manuel Portero-Otín Manuel Portero-Otín Experimental Medicine Department, University of Lleida-IRB Lleida, Lleida, Spain Search for more papers by this author Reinald Pamplona Reinald Pamplona Experimental Medicine Department, University of Lleida-IRB Lleida, Lleida, Spain Search for more papers by this author Isidre Ferrer Isidre Ferrer Department of Pathology and Experimental Therapeutics, Faculty of Medicine, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain Center for Biomedical Research on Neurodegenerative Diseases (CIBERNED), ISCIII, Madrid, Spain Institute of Neurosciences, University of Barcelona, Barcelona, Spain IDIBELL-Bellvitge University Hospital, L'Hospitalet de Llobregat, Spain Search for more papers by this author M Flint Beal M Flint Beal Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY, USA Search for more papers by this author Stéphane Fourcade Corresponding Author Stéphane Fourcade [email protected] orcid.org/0000-0002-8031-5007 Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Search for more papers by this author Aurora Pujol Corresponding Author Aurora Pujol [email protected] orcid.org/0000-0002-9606-0600 Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain Search for more papers by this author Author Information Pablo Ranea-Robles1,2, Nathalie Launay1,2, Montserrat Ruiz1,2, Noel Ylagan Calingasan3, Magali Dumont4, Alba Naudí5, Manuel Portero-Otín5, Reinald Pamplona5, Isidre Ferrer6,7,8,9, M Flint Beal3, Stéphane Fourcade *,1,2 and Aurora Pujol *,1,2,10 1Neurometabolic Diseases Laboratory, Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain 2CIBERER U759, Center for Biomedical Research on Rare Diseases, ISCIII, Barcelona, Spain 3Feil Family Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY, USA 4UMR S 1127, Inserm, U1127, CNRS, UMR 7225, Institut du Cerveau et de la Moelle épinière, Sorbonne Universités, UPMC Université Paris 06, Paris, France 5Experimental Medicine Department, University of Lleida-IRB Lleida, Lleida, Spain 6Department of Pathology and Experimental Therapeutics, Faculty of Medicine, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain 7Center for Biomedical Research on Neurodegenerative Diseases (CIBERNED), ISCIII, Madrid, Spain 8Institute of Neurosciences, University of Barcelona, Barcelona, Spain 9IDIBELL-Bellvitge University Hospital, L'Hospitalet de Llobregat, Spain 10Catalan Institution of Research and Advanced Studies (ICREA), Barcelona, Spain *Corresponding author. Tel: +34 932 60 71 37; Fax: +34 932 60 74 14; E-mail: [email protected] *Corresponding author. Tel: +34 932 60 71 37; Fax: +34 932 60 74 14; E-mail: [email protected] EMBO Mol Med (2018)10:e8604https://doi.org/10.15252/emmm.201708604 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The nuclear factor erythroid 2-like 2 (NRF2) is the master regulator of endogenous antioxidant responses. Oxidative damage is a shared and early-appearing feature in X-linked adrenoleukodystrophy (X-ALD) patients and the mouse model (Abcd1 null mouse). This rare neurometabolic disease is caused by the loss of function of the peroxisomal transporter ABCD1, leading to an accumulation of very long-chain fatty acids and the induction of reactive oxygen species of mitochondrial origin. Here, we identify an impaired NRF2 response caused by aberrant activity of GSK-3β. We find that GSK-3β inhibitors can significantly reactivate the blunted NRF2 response in patients' fibroblasts. In the mouse models (Abcd1− and Abcd1−/Abcd2−/− mice), oral administration of dimethyl fumarate (DMF/BG12/Tecfidera), an NRF2 activator in use for multiple sclerosis, normalized (i) mitochondrial depletion, (ii) bioenergetic failure, (iii) oxidative damage, and (iv) inflammation, highlighting an intricate cross-talk governing energetic and redox homeostasis in X-ALD. Importantly, DMF halted axonal degeneration and locomotor disability suggesting that therapies activating NRF2 hold therapeutic potential for X-ALD and other axonopathies with impaired GSK-3β/NRF2 axis. Synopsis This study reports an aberrant, chronic inhibition of the endogenous antioxidant response driven by NRF2 in adrenoleukodystrophy. Reactivating this pathway in the mouse model with dimethyl fumarate unveils a promising therapeutic option. NRF2-mediated antioxidant response is impaired in X-linked adrenoleukodystrophy (X-ALD) mouse spinal cord and human fibroblasts. Aberrant GSK-3β activity is responsible for the NRF2 blunted response against oxidative stress in X-ALD fibroblasts. Molecular defects present in the X-ALD mouse model are rescued after oral treatment with dimethyl fumarate, an NRF2 activator in current use for multiple sclerosis. Axonal degeneration and locomotor impairment in X-ALD mouse model are prevented with dimethyl fumarate, indicating its therapeutic potential for X-ALD patients. Introduction Oxidative stress and mitochondrial dysfunction contribute to the onset and progression of age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's, Huntington's, and Alzheimer's disease (Lin & Beal, 2006). A common theme among these disorders, as well as the prototypic demyelinating disease multiple sclerosis, is axonal degeneration (Li et al, 2001; Tallantyre et al, 2010). Endogenous antioxidant responses are controlled by nuclear factor erythroid 2-like 2 (NRF2, encoded by NFE2L2), which binds to antioxidant response element (ARE) in the promoter region of target genes, subsequently activating the transcription of genes encoding phase II detoxifying enzymes and cytoprotective defences against oxidative stress (Itoh et al, 1997). These genes include heme oxygenase-1 (HMOX1), NAD(P)H:quinone oxidoreductase-1 (NQO1), and enzymes of glutathione metabolism, such as glutathione S-transferases (GST), glutamate-cysteine ligase (GCL), and glutathione peroxidases (McMahon et al, 2001; Lee et al, 2003). NRF2 also regulates proteostasis (Komatsu et al, 2010; Pajares et al, 2016), neuroinflammation (Innamorato et al, 2008; Rojo et al, 2010), and bioenergetic homeostasis (Holmstrom et al, 2013) in the nervous system, such that activating NRF2-dependent responses initiates a sustained neuroprotective effect in several neurodegenerative disorder models (Kanninen et al, 2009; Neymotin et al, 2011; Stack et al, 2011; Kaidery et al, 2013; Lastres-Becker et al, 2016). We therefore sought to explore the role of NRF2 pathway in the neurodegenerative processes of X-linked adrenoleukodystrophy (X-ALD; McKusick no. 300100). This is the most common peroxisomal disease and leukodystrophy with an incidence of 1:15,000 (Kemper et al, 2017). It is caused by mutations in the ABCD1 gene (Mosser et al, 1993) located on Xq.28, which encodes a peroxisomal transporter that moves very long-chain fatty acids (VCLFA) into the peroxisome for degradation by β-oxidation (van Roermund et al, 2008; Wiesinger et al, 2013). As a consequence, very long-chain fatty acids (VLCFA), especially C26:0, accumulate in tissues and plasma and constitute a pathognomonic biomarker for diagnosis. There are two main forms of the disease (Engelen et al, 2012). First, cerebral adrenoleukodystrophy is present mostly in boys between 5 and 10 years (35–40% of the cases) but also in adolescents and adult men, who present a strong inflammatory demyelinating reaction in central nervous system white matter. Second, adrenomyeloneuropathy occurs in 60% of the cases and affects adult men and heterozygous women over the age of 40 (Engelen et al, 2014). Adrenomyeloneuropathy is characterized by peripheral neuropathy and distal axonopathy involving corticospinal tracts of the spinal cord. The clinical presentation of X-ALD varies even in the same family, which suggests the presence of modifier genes or environmental factors (Berger et al, 1994; Turk et al, 2017). Current therapeutic options are restricted to bone marrow transplantation (Miller et al, 2011) and hematopoietic stem cell gene therapy (Cartier et al, 2009; Eichler et al, 2017), and are limited by a very narrow therapeutic window, which reinforces the need to develop additional therapies for this devastating disease. The mouse model of X-ALD (Abcd1− mice) develops axonopathy and locomotor impairment very late in life, at 20 months of age, resembling adrenomyeloneuropathy, the most frequent X-ALD phenotype (Pujol et al, 2002). The closest homolog Abcd2 exhibits overlapping metabolic functions (Fourcade et al, 2009) and has been postulated as modifier of the biochemical defect (Muneer et al, 2014). Double mutant Abcd1−/Abcd2−/− mice develop a more severe, earlier onset axonopathy starting at 12 months of age, what makes them a more suitable model for therapeutic essays (Pujol et al, 2002; Mastroeni et al, 2009; Lopez-Erauskin et al, 2011; Morato et al, 2013, 2015; Launay et al, 2015, 2017). Using these mouse models and patients' samples, studies by our laboratory and others have revealed that VLCFA-induced oxidative stress is a critical, early pathogenic factor in X-ALD (Vargas et al, 2004; Powers et al, 2005; Fourcade et al, 2008, 2010; Hein et al, 2008; Lopez-Erauskin et al, 2011; Petrillo et al, 2013), although the exact mechanisms by which VLCFA-induced redox imbalance causes neurodegeneration in X-ALD remain unclear (Fourcade et al, 2015). Here, we examined whether the NRF2 antioxidant pathway could contribute to the increased oxidative damage detected in this disease, in both the Abcd1− mouse model and the skin fibroblasts derived from X-ALD patients. We also treated X-ALD mouse models (Abcd1− and Abcd1−/Abcd2−/− mice) with dimethyl fumarate (DMF, BG-12, Tecfidera), an NRF2 activator (Linker et al, 2011; Scannevin et al, 2012), that is a currently approved medication for relapsing-remitting multiple sclerosis (Fox et al, 2012; Gold et al, 2012). Results GSK-3β/NRF2 antioxidant pathway is altered in Abcd1− mice We previously identified a redox dyshomeostasis in X-ALD, characterized by an excess of reactive oxygen species (ROS) production and repression of key antioxidant enzymes (Fourcade et al, 2008). Since NRF2 plays a critical role in the antioxidant cellular defence, we asked whether the NRF2-dependent antioxidant pathway was altered in the Abcd1 null mouse. We found decreased NRF2 protein levels in Abcd1− mice spinal cord at 12 months of age (Fig 1A), a presymptomatic disease stage in this mouse model. Dysregulated NRF2 protein levels were organ-specific, as we did not observe any changes in non-affected tissues in the mouse model, such as cerebral cortex or liver (Fig EV1). To verify that lower protein levels had functional consequences, we measured mRNA expression of NRF2 classical target genes (Hmox1, Nqo1 and glutathione S-transferase alpha-3, Gsta3) at the same age. We observed a slight but significant decreased expression of these three NRF2 target genes in the Abcd1− mouse spinal cord at 12 months of age (Fig 1B), consistent with a downregulated NRF2 pathway. Figure 1. Altered GSK-3β/NRF2 antioxidant pathway in Abcd1− mice A. Representative immunoblot of NRF2 protein level measured in WT (n = 6) and Abcd1− (n = 6) mice spinal cord at 12 months of age. Protein levels normalized relative to γ-tubulin (γ-TUB) and quantification depicted as fold change to WT mice. B. NRF2-dependent antioxidant gene expression (Hmox1, Nqo1, and Gsta3) in WT (n = 8) and Abcd1− (n = 8) mice spinal cord at 12 months of age. Gene expression normalized relative to mouse Rplp0 and depicted as fold change to WT mice. C, D. Representative immunoblots of pSer473 AKT, pThr308 AKT, AKT, pSer9 GSK-3β, pTyr216 GSK-3β, and GSK-3β protein level in WT (n = 12) and Abcd1− (n = 12) mice spinal cord at 12 months of age. Protein level normalized relative to corresponding non-phosphorylated proteins or γ-TUB (in the case of AKT and GSK-3β). Quantification depicted as fold change to WT mice. Data information: In (A, B, and D), data are presented as mean ± SD. *P < 0.05 (unpaired Student's t-test). See the exact P-values in Appendix Table S3. Source data are available online for this figure. Source Data for Figure 1 [emmm201708604-sup-0003-SDataFig1.tif] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Basal NRF2 levels in other mice tissues and human fibroblasts A. Representative immunoblots of NRF2 protein level in WT (n = 8) and Abcd1− (n = 8) mice cerebral cortex and liver at 12 months of age. Protein levels normalized relative to γ-tubulin (γ-TUB) and quantification depicted as fold change to WT mice. B. Representative immunoblots of NRF2 protein level in total extracts from CTL (n = 5) and X-ALD (n = 5) fibroblasts. Protein levels normalized relative to γ-TUB. Quantification is depicted as fold change to control fibroblasts. Data information: Data are presented as mean ± SD. Download figure Download PowerPoint Several signals can regulate NRF2-dependent responses, in particular those that modulate GSK-3β activity (Salazar et al, 2006; Rojo et al, 2008; Rada et al, 2011). We thus examined the activity of the AKT/GSK-3β pathway in the spinal cord of Abcd1− mice by measuring the phosphorylation of serine 473 (pSer473) and threonine 308 (pThr308) residues of AKT, which reflects its activation. We also measured the phosphorylation of serine 9 (pSer9) and tyrosine 216 (pTyr216) residues of GSK-3β, which indicate inhibition or activation of GSK-3β, respectively. We found less AKT activation in Abcd1− mice spinal cord, as shown by decreased pThr308 AKT relative to total AKT levels. Defective AKT phosphorylation resulted in the activation of GSK-3β, indicated by reduced pSer9 GSK-3β compared with total GSK-3β levels (Fig 1C and D). We did not observe any changes in pSer473, pTyr216, or in the total levels of AKT and GSK-3β (Fig 1C and D). These data indicate a dysregulated AKT/GSK-3β/NRF2 axis in the Abcd1− mouse spinal cord, with predicted higher activity of GSK-3β upstream of NRF2. Impaired NRF2-dependent antioxidant pathway is mediated by GSK-3β in patients' fibroblasts Primary fibroblasts from X-ALD patients provide a good surrogate cell model to dissect disease mechanisms, as they recapitulate the main disease hallmarks: accumulation of VLCFA (Moser et al, 1980), higher production of free radicals of mitochondrial origin (Lopez-Erauskin et al, 2013), loss of energetic homeostasis (Galino et al, 2011), altered proteostasis (Launay et al, 2013, 2015), and endoplasmic reticulum (ER) stress (van de Beek et al, 2017; Launay et al, 2017). Using this cell system, we determined whether patients' fibroblasts exhibited an altered AKT/GSK-3β/NRF2 pathway. At baseline, we observed equivalent NRF2 protein levels in patients' fibroblasts compared with controls (Fig EV1). We then tested the functionality of the NRF2 pathway, by treating patients' and control fibroblasts either with C26:0, the primary VLCFA accumulated in patients, or with oligomycin, which acts as a generator of mitochondrial ROS inhibiting complex V (Fourcade et al, 2008; Paupe et al, 2009). Both compounds produce mitochondrial ROS in these fibroblasts (Lopez-Erauskin et al, 2013). We show that both C26:0 and oligomycin activated NRF2-dependent responses in control fibroblasts, characterized by both higher NRF2 translocation to the nucleus (Fig 2A and B) and increased expression of NRF2 target genes (HMOX1, NQO1, and GCLC mRNA; Fig 2C). However, this physiological response against oxidative stress was blunted in X-ALD fibroblasts with both ROS-producing stimuli (Fig 2A–C). Figure 2. Impaired AKT/GSK-3β/NRF2 antioxidant response after oxidative stress in X-ALD patients' fibroblasts A, B. Representative immunoblots of NRF2 protein translocation to the nucleus upon VLCFA (C26:0, 50 μM, 24 h) or oligomycin (15 μM, 18 h) in control (CTL, n = 5 per condition, left panels) and X-ALD (n = 5 per condition, right panels) fibroblasts. Protein levels normalized relative to lamin B1 in the nuclear fraction, aldolase A (ALDOA) in the cytoplasmic fraction, and γ-TUB in the total fraction. Quantification depicted as fold change to vehicle-treated (Veh) fibroblasts. C. NRF2-dependent antioxidant gene expression (HMOX1, NQO1, and GCLC) upon oxidative stress in CTL (n = 5 per condition) and X-ALD (n = 5 per condition) fibroblasts. Gene expression normalized relative to RPLP0. Quantification depicted as fold change to vehicle-treated (Veh) fibroblasts. D, E. Representative immunoblots of pSer473 AKT, pThr308 AKT, AKT, pSer9 GSK-3β, and GSK-3β measured after oxidative stress in CTL (n = 5 per condition) and X-ALD (n = 5 per condition) fibroblasts. Protein levels normalized relative to corresponding non-phosphorylated proteins or γ-TUB (in the case of AKT and GSK-3β). Quantification depicted as fold change to vehicle-treated (Veh) fibroblasts. F. NRF2-dependent antioxidant gene expression (HMOX1, NQO1, and GCLC) after GSK-3β inhibition in VLCFA-treated CTL (n = 8 per condition) and X-ALD (n = 8 per condition) fibroblasts. Gene expression normalized relative to RPLP0. Quantification depicted as fold change to vehicle-treated (Veh) fibroblasts. Data information: In (B, C, E, and F), data are presented as mean ± SD. In (B, E, and F), *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Tukey's post hoc test). In (C), #P < 0.05, ##P < 0.01, ###P < 0.001 (one-way ANOVA followed by Dunnett's post hoc test). In (C and F), $P < 0.01, $$P < 0.01 (non-parametric Kruskal–Wallis' test followed by Dunn's post hoc test). See the exact P-values in Appendix Table S3. Source data are available online for this figure. Source Data for Figure 2 [emmm201708604-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Moreover, both treatments elicited AKT activation (increased pSer473 and pThr308) and subsequent GSK-3β inactivation (higher pSer9 GSK-3β levels) in control fibroblasts (Fig 2D and E). Again, this physiological response against oxidative stress was impaired in X-ALD fibroblasts, as phosphorylated levels of AKT and GSK-3β did not change following C26:0 or oligomycin treatment (Fig 2D and E). As GSK-3β activation can repress NRF2, we sought to determine whether this phenomenon was interrelated in the cellular model. For this, we assessed whether specific GSK-3β inhibitors (CT99021 and SB216763; Coghlan et al, 2000; Ring et al, 2003) could restore a normal NRF2-dependent response in X-ALD fibroblasts. Indeed, treatment with both compounds reactivated the NRF2 pathway, characterized by an upregulation of the NRF2-target genes HMOX1, NQO1, and GCLC in patients' fibroblasts upon incubation with excess of C26:0 (Fig 2F). Collectively, these data indicate that the aberrant GSK-3β activation upstream of NRF2 governs the blunted NRF2-dependent response upon oxidative stress in this disease model. DMF rescues mitochondrial depletion, bioenergetic failure, and oxidative damage in Abcd1− mice To elucidate the impact of a defective NRF2-dependent response in the pathogenesis of adrenoleukodystrophy, we decided to treat Abcd1− mice with DMF, a classical activator of NRF2 (Linker et al, 2011; Scannevin et al, 2012). Dimethyl fumarate has therapeutic efficacy for relapsing-remitting multiple sclerosis (Fox et al, 2012; Gold et al, 2012) and besides, preclinical tests show success to treat other neurodegenerative diseases like Huntington's (Ellrichmann et al, 2011) and Parkinson's disease (Ahuja et al, 2016; Lastres-Becker et al, 2016). Before treating the animals, we tested DMF in control and X-ALD fibroblasts. We found that DMF reactivated the NRF2-blunted response upon VLCFA addition (Fig EV2), similar to the GSK-3β inhibitors used (Fig 2F). Moreover, DMF alone induced HMOX1 and NQO1 expression in control fibroblasts and also HMOX1 expression in X-ALD fibroblasts (Fig EV2). Thus, these new data reinforced the rational for DMF treatment in vivo. Click here to expand this figure. Figure EV2. DMF effect on NRF2 response in X-ALD fibroblastsExpression of NRF2 target genes was measured in CTL (n = 3) and X-ALD (n = 3) after C26:0 (50 μM, 24 h) and/or DMF (20 μM, 6 h). Gene expression normalized relative to RPLP0. Quantification depicted as fold change to vehicle-treated (Veh) fibroblasts. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Tukey's post hoc test). See the exact P-values in Appendix Table S3. Download figure Download PowerPoint We fed Abcd1− mice with DMF-containing chow at 100 mg/kg, starting at 8 months of age, for 4 months. First, we verified the efficacy of dietary DMF administration by measuring NRF2 protein levels and mRNA expression of three classical NRF2-target genes (Hmox1, Nqo1, and Gsta3). Dimethyl fumarate treatment rescued both NRF2 protein levels (Fig 3A) and NRF2 targets in Abcd1− mice spinal cord at 12 months of age (Fig 3B). Figure 3. NRF2 activation by DMF prevents oxidative damage to proteins and lipids, mitochondrial depletion, and bioenergetic failure in Abcd1− mice A. Representative immunoblot of NRF2 protein levels in WT (n = 6), Abcd1− (n = 6) and DMF-treated Abcd1− mice (Abcd1− + DMF, n = 6) mice spinal cord at 12 months of age. Protein levels normalized relative to γ-TUB. Quantification depicted as fold change to WT mice. B. NRF2-dependent antioxidant gene expression (Hmox1, Nqo1, and Gsta3) in WT (n = 8), Abcd1− (n = 8), and Abcd1− + DMF (n = 8) mice spinal cord at 12 months of age. Gene expression normalized relative to Rplp0. Quantification represented as fold change to WT mice. C. Oxidative lesions to lipids and proteins in WT (n = 5), Abcd1− (n = 5), and Abcd1− + DMF (n = 5) mice spinal cord at 12 months of age. AASA, CEL, CML, and MDAL levels measured by GC/MS. Quantification represented as fold change to WT mice. D. mtDNA levels in WT (n = 8), Abcd1− (n = 8), and Abcd1− + DMF (n = 8) mice spinal cord at 12 months of age. mtDNA content expressed as the ratio of mtDNA (CytB levels) to nDNA (Cebpa levels). Quantification depicted as fold change to WT mice. E. Sirt1, Ppargc1a, Nrf1, and Tfam gene expression in WT (n = 8), Abcd1− (n = 8), and Abcd1− + DMF (n = 8) mice spinal cord at 12 months of age. Gene expression normalized relative to Rplp0. Quantification depicted as fold change to WT mice. F. ATP levels in WT (n = 8), Abcd1− (n = 8), and Abcd1− + DMF (n = 8) mice spinal cord at 12 months of age. Quantification represented as fold change to WT mice. Data information: Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Tukey's post hoc test). In (B), #P < 0.05 (one-way ANOVA followed by Dunnett's post hoc test). In (E), $P < 0.05 (non-parametric Kruskal–Wallis' test followed by Dunn's post hoc test). See the exact P-values in Appendix Table S3. Source data are available online for this figure. Source Data for Figure 3 [emmm201708604-sup-0005-SDataFig3.tif] Download figure Download PowerPoint Next, we measured the effect of DMF on several quantitative markers of oxidative damage to lipids and proteins, such as direct carbonylation of proteins (Aminoadipic semialdehyde: AASA), glycoxidation (Nɛ-(carboxyethyl)-lysine: CEL and Nɛ-(carboxymethyl)-lysine: CML), and protein lipoxidation (Nɛ-malondialdehyde-lysine: MDAL; Fourcade et al, 2008). We found an antioxidant role for DMF in this model, as it normalized AASA, CEL, CML, and MDAL in Abcd1− mice spinal cord (Fig 3C). We also examined the effect of DMF on mitochondrial dysfunction (Morato et al, 2013, 2015). DMF normalized mitochondrial biogenesis, based on different parameters: mtDNA levels (Fig 3D) and mRNA expression of sirtuin-1, Sirt1; peroxisome proliferator-activated receptor gamma coactivator 1-alpha, Ppargc1a; nuclear respiratory factor-1, Nrf1; and transcription factor A, mitochondrial, Tfam (Fig 3E; Morato et al, 2013, 2015). We previously reported decreased levels of ATP in the spinal cord of Abcd1− mice (Galino et al, 2011), suggesting that deficient energy homeostasis is a key feature in X-ALD pathology. In
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