Oxidative damage compromises energy metabolism in the axonal degeneration mouse model of X-adrenoleukodystrophy

Abstract

Aims: Chronic metabolic impairment and oxidative stress are associated with the pathogenesis of axonal dysfunction in a growing number of neurodegenerative conditions. To investigate the intertwining of both noxious factors, we have chosen the mouse model of adrenoleukodystrophy (X-ALD), which exhibits axonal degeneration in spinal cords and motor disability. The disease is caused by loss of function of the ABCD1 transporter, involved in the import and degradation of very long-chain fatty acids (VLCFA) in peroxisomes. Oxidative stress due to VLCFA excess appears early in the neurodegenerative cascade.

Results: In this study, we demonstrate by redox proteomics that oxidative damage to proteins specifically affects five key enzymes of glycolysis and TCA (Tricarboxylic acid) cycle in spinal cords of Abcd1(-) mice and pyruvate kinase in human X-ALD fibroblasts. We also show that NADH and ATP levels are significantly diminished in these samples, together with decrease of pyruvate kinase activities and GSH levels, and increase of NADPH.

Innovation: Treating Abcd1(-) mice with the antioxidants N-acetylcysteine and α-lipoic acid (LA) prevents protein oxidation; preserves NADH, NADPH, ATP, and GSH levels; and normalizes pyruvate kinase activity, which implies that oxidative stress provoked by VLCFA results in bioenergetic dysfunction, at a presymptomatic stage.

Conclusion: Our results provide mechanistic insight into the beneficial effects of antioxidants and enhance the rationale for translation into clinical trials for X-adrenoleukodystrophy.

FIG. 1.

ALDO A, PGK1, PKM2, DLD, and ACO2 are more highly oxidized in spinal cord from 12 month-old Abcd1⁻ mice. (A) Redox proteomics experiments in Wt and Abcd1⁻ mice. Western blot with an antibody anti-DNP was performed to identify oxidized proteins (n=5/genotype). A validation Western blot was performed with specific antibodies against aldolase A (ALDO A), phosphoglycerate kinase (PGK1), pyruvate kinase (PKM2), dihydrolipoamide dehydrogenase (DLD), and mitochondrial aconitase (ACO2) after identification obtained by MS (B), allowing relative quantification of their expression (C). Relative protein level is expressed as a percentage of control, and referred to γ-tubulin as loading marker. (D) Pkm2 was quantified by Q-PCR in Wt and Abcd1⁻ mice. 36b4 was used as internal control (n=10–12 by genotype). Statistical analysis was done by Student's t-test: *p<0.05.

FIG. 2.

PKM2 is oxidized by C26:0 excess in human fibroblasts. (A) Redox proteomics experiments were performed in human control and X-ALD fibroblasts (n=5 per genotype and condition), which were treated for 7 days with BSA-conjugated C26:0 (100 μM) or BSA as control in a serum-free medium. Western blot with an antibody anti-DNP was performed to identify oxidized proteins. Western blot was performed with a specific antibody against pyruvate kinase (PKM2) to validate the protein identification obtained by MS (B) or to quantify their expression (C). Relative protein level is expressed as a percentage of control, and referred to γ-tubulin as loading marker. Significant differences were revealed by ANOVA followed by Tukey HSD post hoc test.

FIG. 3.

Metabolite levels in 12 month-old spinal cord from Abcd1-deficient mice. (A) Fructose 1–6 bisphosphate, dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3 phosphate (GA3P), 2 and 3-phosphoglycerate, pyruvate, lactate, oxalacetate, citrate, α-ketoglutarate, fumarate, and malate levels in Wt and Abcd1⁻ mice. (B) Pyruvate/lactate ratio is not modified in Abcd1⁻ spinal cord at 12m of age. (C) Pyruvate kinase activity is decreased in 12 month Abcd1-null mice spinal cord. Pyruvate kinase activity is expressed as units/mg tissue (n=5–7/genotype). Statistical analysis was done with Student's t-test (*p≤0.05, **p≤0.01, and ***p≤0.001).

FIG. 4.

ABCD1 loss disturbs NADH, NADPH, GSH, and ATP levels in spinal cord from 12 month-old Abcd1-null mice. (A) NADH, NAD⁺ levels and NAD⁺/NADH ratio, (B) NADPH, NADP⁺ levels and NADP⁺/NAPH, (C) GSH levels, and (D) ATP levels in Wt and Abcd1-null mice (n=8/genotype). Statistical analysis was done with Student's t-test: *p<0.05, **p<0.01, ***p<0.001.

FIG. 5.

Metabolic failure is prevented by a combination of antioxidants. (A) Redox proteomics experiments were performed in 12 month-old Abcd1⁻ and Abcd1⁻ mice fed for 4 months (Abcd1⁻+Antx) with NAC and LA. Western blot with an antibody anti-DNP was performed to identify oxidized proteins (n=5/genotype). (B) Western blot (n=4/genotype) against aldolase A (ALDO A), phosphoglycerate kinase (PGK1), pyruvate kinase (PKM2), dihydrolipoamide dehydrogenase (DLD), and mitochondrial aconitase (ACO2). Pyruvate kinase expression (C) and activity (D), NADH, NAD⁺ levels and the NAD⁺/NADH ratio (E), NADPH, NADP⁺ levels and NADP⁺/NADPH ratio (F), GSH levels, (G) and ATP levels (H) were measured in spinal cord from 12 month-old Wt, Abcd1⁻ and Abcd1⁻ mice fed for 4 months with a cocktail of antioxidants (Abcd1⁻+Antx) (n=6–7 mice per genotype and condition). Statistical analysis was done with ANOVA followed by Tukey HSD post hoc test. Significant differences are shown as *p<0.05, **p<0.01 and ***p<0.001.

FIG. 6.

Working hypothesis on the interplay between metabolic failure and oxidative stress in X-ALD. C26:0 excess generates ROS, which results in oxidation of enzymes belonging to glycolysis and TCA cycle. This oxidative damage provokes a reduction in enzyme activities, which is demonstrated by alteration of the substrate and/or product concentrations of these enzymes. An impairment of TCA leads to a reduction in NADH level, the substrate of complex I of the mitochondria respiratory chain, contributing to decreased production of ATP. This ignites a vicious circle increasing ROS production. Then, the inhibition of complex I leads to a reduction in ATP production by the mitochondria respiratory chain. Combination of NAC and LA prevent metabolic failure by protecting key enzymes of TCA and glycolysis from oxidation. Further, lipoic acid might ameliorate the function of KGDHC and PDHC.