Pioglitazone halts axonal degeneration in a mouse model of X-linked adrenoleukodystrophy

Abstract

X-linked adrenoleukodystrophy is a neurometabolic disorder caused by inactivation of the peroxisomal ABCD1 transporter of very long-chain fatty acids. In mice, ABCD1 loss causes late onset axonal degeneration in the spinal cord in association with locomotor disability resembling the most common phenotype in patients, adrenomyeloneuropathy. Increasing evidence indicates that oxidative stress and bioenergetic failure play major roles in the pathogenesis of X-linked adrenoleukodystrophy. In this study, we aimed to evaluate whether mitochondrial biogenesis is affected in X-linked adrenoleukodystrophy. We demonstrated that Abcd1 null mice show reduced mitochondrial DNA concomitant with downregulation of mitochondrial biogenesis pathway driven by PGC-1α/PPARγ and reduced expression of mitochondrial proteins cytochrome c, NDUFB8 and VDAC. Moreover, we show that the oral administration of pioglitazone, an agonist of PPARγ, restored mitochondrial content and expression of master regulators of biogenesis, neutralized oxidative damage to proteins and DNA, and reversed bioenergetic failure in terms of ATP levels, NAD+/NADH ratios, pyruvate kinase and glutathione reductase activities. Most importantly, the treatment halted locomotor disability and axonal damage in X-linked adrenoleukodystrophy mice. These results lend support to the use of pioglitazone in clinical trials with patients with adrenomyeloneuropathy and reveal novel molecular mechanisms of action of pioglitazone in neurodegeneration. Future studies should address the effects of this anti-diabetic drug on other axonopathies in which oxidative stress and mitochondrial dysfunction are contributing factors.

Keywords: X-linked adrenoleukodystrophy; axonal degeneration; mitochondrial biogenesis; oxidative stress; pioglitazone.

Figure 1

Mitochondrial DNA and mitochondrial protein levels are reduced in spinal cords of Abcd1^−/− mice at 12 months of age. Wild-type (n = 8) and Abcd1^−/− (n = 8) mice at 12 months of age. (A) NDUFB8 and VDAC protein levels, (B) mitochondrial DNA (mtDNA) levels, (C) cytochrome c quantification by immunostaining in mice spinal cord histological slides and (D) relative gene expression of PGC-1α, PGC-1β, PPARα, PPARβ, PPARγ, ERRα, NRF1 and TFAM. Scale bar = 100 µm (low magnification) / 20 µm (high magnification). Data represent mean ± SD (*P ≤ 0.05, **P ≤ 0.01). WT = wild-type.

Figure 2

Mitochondrial DNA and mitochondrial protein levels are reduced in the affected white matter of patients with X-linked adrenoleukodystrophy. Affected (n = 4) and normal-appearing white matter (n = 4) of patients with X-linked adrenoleukodystrophy. (A) Mitochondrial DNA (mtDNA) levels and (B) NDUFB8 and VDAC protein levels. Data represent mean ± SD (*P ≤ 0.05, **P ≤ 0.01).

Figure 3

Pioglitazone normalizes mitochondrial DNA and mitochondrial protein levels in spinal cords of Abcd1^−/− mice. Wild-type (n = 8), Abcd1^−/− (n = 8) and Abcd1^−/− mice fed for 2 months with pioglitazone (Abcd1⁻ + PIO) (n = 8) at 12 months of age. (A) Relative gene expression of iNOS and COX2, (B) mitochondrial DNA (mtDNA) level, (C) NDUFB8 and VDAC protein levels and (D) relative gene expression of PGC-1α, NRF1, TFAM and PPARα, PPARβ and PPARγ. Data represent mean ± SD (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). WT = wild-type.

Figure 4

Pioglitazone normalizes ATP, NADH levels and pyruvate kinase in spinal cords of Abcd1 null mice. Wild-type (n = 8), Abcd1^−/− (n = 8) and Abcd1^−/− mice fed for 2 months with pioglitazone (Abcd1⁻ + PIO) (n = 8) at 12 months of age. (A) ATP, (B) NAD⁺ and NADH levels, (C) pyruvate kinase activity and (D) pyruvate kinase protein levels. Data represent mean ± SD (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). WT = wild-type.

Figure 5

Pioglitazone reverses oxidative lesions in spinal cords of Abcd1^−/− mice. Wild-type (n = 8), Abcd1^−/− (n = 8) and Abcd1^−/− mice fed for 2 months with pioglitazone (Abcd1⁻ + PIO) (n = 8) at 12 months of age. (A) GSA, AASA, CML and MDAL levels. (B) SOD2 and GPX1 protein levels. (C) Glutathione reductase (GR) activity and protein levels. Data represent mean ± SD (*P ≤ 0.05).

Figure 6

Oxidative stress, myelin and axonal pathologies and mitochondrial depletion are prevented by pioglitazone in spinal cords of 18-month-old Abcd1^−/−/Abcd2^−/− mice. Longitudinal and transversal sections of the spinal cord in wild-type (A, D, G, J, M, P, S, V), Abcd1^−/−/Abcd2^−/− (DKO) (B, E, H, K, N, Q, T, W) and Abcd1^−/−/Abcd2^−/− + PIO (DKO + PIO) (C, F, I, L, O, R, U, X) mice processed for 8-oxodG (A–C), lectin Lycopericon esculentum (D–F), GFAP (G–I), synaptophysin (J–L), APP (M–O), Sudan black (P–R), cytochrome c (S–U), and SMI-32 (V–X). Scale bar = 100 µm. (Y) Quantification of amyloid precursor protein (APP) and synaptophysin accumulation in axonal swellings in wild-type (WT), double knockout (DKO) and double knockout + PIO (DKO + PIO) mice. Significant differences were determined as described in the ‘Materials and methods’ section (n = 5–6 mice per genotype and condition; *P < 0.05, **P < 0.01, ***P < 0.001). DKO = double knockout.

Figure 7

Pioglitazone improves locomotor disability in an X-linked adrenoleukodystrophy mouse model. (A) Treadmill and (B) bar-cross test in wild-type (WT) and Abcd1^−/−/Abcd2^−/− mice (DKO) mice at 13 months of age (before treatment) and after 2 and 4 months (mo) of pioglitazone treatment (DKO + PIO). Data represent mean ± SD. Statistical analysis was carried out with Student’s t-test (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001) or ANOVA followed by Tukey HSD post hoc (wild-type versus DKO = *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; DKO versus DKO + PIO = ^#P ≤ 0.05, ^##P ≤ 0.01). DKO = double knockout.