Antioxidants halt axonal degeneration in a mouse model of X-adrenoleukodystrophy

Abstract

Objective: Axonal degeneration is a main contributor to disability in progressive neurodegenerative diseases in which oxidative stress is often identified as a pathogenic factor. We aim to demonstrate that antioxidants are able to improve axonal degeneration and locomotor deficits in a mouse model of X-adrenoleukodystrophy (X-ALD).

Methods: X-ALD is a lethal disease caused by loss of function of the ABCD1 peroxisomal transporter of very long chain fatty acids (VLCFA). The mouse model for X-ALD exhibits a late onset neurological phenotype with locomotor disability and axonal degeneration in spinal cord resembling the most common phenotype of the disease, adrenomyeloneuropathy (X-AMN). Recently, we identified oxidative damage as an early event in life, and the excess of VLCFA as a generator of radical oxygen species (ROS) and oxidative damage to proteins in X-ALD.

Results: Here, we prove the capability of the antioxidants N-acetyl-cysteine, α-lipoic acid, and α-tocopherol to scavenge VLCFA-dependent ROS generation in vitro. Furthermore, in a preclinical setting, the cocktail of the 3 compounds reversed: (1) oxidative stress and lesions to proteins, (2) immunohistological signs of axonal degeneration, and (3) locomotor impairment in bar cross and treadmill tests.

Interpretation: We have established a direct link between oxidative stress and axonal damage in a mouse model of neurodegenerative disease. This conceptual proof of oxidative stress as a major disease-driving factor in X-AMN warrants translation into clinical trials for X-AMN, and invites assessment of antioxidant strategies in axonopathies in which oxidative damage might be a contributing factor.

Figure 1

Trolox, N-acetylcysteine and α-lipoic acid (LA) prevent radical oxygen species (ROS) generated by C26:0. Intracellular ROS was measured in control (n = 5) and X-adrenoleukodystrophy (X-ALD) human fibroblasts (n = 5) after 24 hours. Three different antioxidants were used at high doses: (A) Trolox, (B) N-acetylcysteine (NAC), and (C) LA. (D) The 3 antioxidants were used alone or in combination at lower doses. Significant differences were determined as described in Materials and Methods (*p < 0.05, **p < 0.01, ***p < 0.001). EtOH = ethyl alcohol; ANTX = antioxidants.

Figure 2

Antioxidant treatment normalizes oxidative lesions markers in spinal cord from 22-month-old Abcd1⁻ mice. (A) Dinitrophenol (DNP) levels in 22-month-old wild-type (Wt), Abcd1⁻, and Abcd1⁻ + antioxidants (Antx) mice. The quantification of these blots by densitometry was performed and normalized to γ-tubulin. (B) N^ɛ-(carboxymethyl)-lysine (CML), N^ɛ-(carboxyethyl)-lysine (CEL), and N^ɛ-malondialdehyde-lysine (MDAL) in Wt, Abcd1⁻, and Abcd1⁻ + Antx mice. (C) GPX1 levels were quantified in Wt, Abcd1⁻, and Abcd1⁻ + Antx mice. Significant differences were determined as described in Materials and Methods (n = 6 mice per genotype and condition; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

Oxidative stress, myelin, and axonal pathologies in 22-month-old Abcd1⁻ spinal cord are prevented by an antioxidant cocktail. Longitudinal sections of the dorsal spinal cord in wild-type (Wt) (A, D, G, J, M, P), Abcd1⁻ (B, E, H, K, N, Q) and Abcd1⁻ + antioxidants (Antx) (C, F, I, L, O, R) mice were processed for 8-oxo-7,8-dihydro-2′-deoxyguanosine marker (8-oxodG) (A–C), lectin Lycopersicon esculentum (D-F), glial fibrillary acidic protein (GFAP) (G–I), synaptophysin (J–L), amyloid precursor protein (APP) (M–O), and Sudan black (P–R). Bar = 25μm. (S) Quantification of APP and synaptophysin accumulation in axonal swellings in Wt, Abcd1⁻, and Abcd1⁻ + Antx mice. Significant differences were determined as described in Materials and Methods (n = 5–6 mice per genotype and condition; **p < 0.01).

Figure 4

A combination of antioxidants rescues locomotor deficits in Abcd1⁻/Abcd2^−/− mice. Bar cross test (A–D) and treadmill experiment (E–I) were carried out at 12 and 18 months of age (wild-type [Wt; n = 12], Wt + antioxidants [Antx; n = 9], Abcd1⁻/Abcd2^−/− [Dko; n = 17], and Abcd1⁻/Abcd2^−/− + Antx [Dko + Antx; n = 12]). The time spent to cross the bar (A, C) and the number of slips (B, D) were quantified at 12 (A, B) and 18 months of age (C, D). Treadmill experiments were performed in Wt and Dko mice at 12 (E, F) and 18 months of age (G, I). Number of shocks (E, H) and latency to falling off the belt (time of shocks in seconds) (F, I) were quantified after 7 minutes. The percentage of mice still running/minute is represented panel G. Significant differences were determined as described in Materials and Methods (*p < 0.05, **p < 0.01, ***p < 0.001).