(M1430-05-29) Exposure-Response Analysis of Nervonic Acid and C26:0-Lysophosphatidylcholine in an Adrenoleukodystrophy Mouse Model - Therapeutic Implications

Purpose: Adrenoleukodystrophy (ALD) is a devastating, rare neurological disorder caused by a mutation in the ABCD1 gene and characterized by the accumulation of saturated very long-chain fatty acids (VLCFA), mainly hexacosanoic acid (C26:0), particularly in plasma, cerebral white matter, spinal cord, adrenal cortex, and testis¹. The accumulation of VLCFA, resulting from impaired peroxisomal β-oxidation (the biochemical defect underlying the disease pathology), leads to adrenal insufficiency, progressive axonal degeneration, and cerebral inflammatory demyelination². Moreover, VLCFA are incorporated into various lipid classes, among which C26:0-lysophosphatidylcholine (C26:0-LPC) levels have been found to correlate with disease severity³. Dietary monounsaturated fatty acids can reduce VLCFA accumulation in patient-derived fibroblasts⁴. Nervonic acid (NA), an essential monounsaturated fatty acid involved in the development and maintenance of the myelin sheath, is emerging as a promising therapeutic candidate for ALD. We have found that NA can effectively decrease C26:0 and total saturated VLCFA levels in ALD fibroblasts⁵. In this study, we conducted a 4-week dietary intervention using two dose levels of NA to investigate its therapeutic potential in modulating disease-relevant lipid biomarkers. The primary objective was to evaluate the efficacy of NA in reducing C26:0-LPC levels in plasma and tissues, and to characterize the exposure-response relationship.

Methods: The Abcd1 knockout mouse model used in this study was developed by the Kennedy Krieger Institute and exhibits reduced β-oxidation and elevated tissue VLCFA levels. Male Abcd1 -/- mice (n=4 per group) aged at 8 weeks old were fed with standard chow, 0.6% NA chow, 3% NA chow, or 3% erucic acid chow (a positive comparator previously shown to reduce VLCFA levels) for 4 weeks. Blood samples were collected weekly to quantify changes in C26:0-LPC levels. Additionally, total lipid concentrations in plasma were determined both before treatment and at the end of treatment (assuming steady-state concentrations at the endpoint). Following a 4-week intervention, target tissues including brain, heart, liver, kidney, and adrenal gland were collected for lipid analysis. The C26:0-LPC in plasma and tissues were analyzed using LC-MS/MS method, whereas the fatty acid concentrations in plasma and tissues were analyzed using GC/MS method. Statistical analyses and exposure-response plot generation were performed using R software (version 4.1.1). A schematic illustration of the study design is shown in Fig. 1.

Results: Both doses of NA significantly reduced plasma C26:0-LPC levels as early as one week after treatment initiation, with the reduction sustained throughout the 4-week intervention. The 3% NA group exhibited a more substantial decrease in plasma C26:0-LPC (~4-fold reduction) compared to the 0.6% NA group (~1.5-fold reduction), indicating a dose-dependent effect of NA in this mouse model. In addition to plasma, C26:0-LPC levels were also significantly reduced in brain tissue following treatment with both doses, compared to the control group. Consistent with the plasma findings, the 3% NA dose led to a greater reduction in brain C26:0-LPC levels than the lower dose, further supporting the dose-dependent therapeutic effect of NA.

Conclusion: In summary, we evaluated the short-term effects of NA on a lipidomic biomarker of ALD using a preclinical mouse model. Our study demonstrated that NA significantly reduced C26:0-LPC levels in both plasma and brain tissue in a dose-dependent manner, with measurable effects observed as early as one week into treatment. These findings suggest that NA effectively targets the VLCFA pathology and holds the potential to prevent or delay disease onset. Overall, this study provides preclinical evidence supporting NA as a promising therapeutic candidate for ALD and lays the groundwork for future investigations into its long-term efficacy, mechanism of action, and potential clinical translation.

References: [1] Kemp S, Valianpour F, Denis S, et al. Elongation of very long-chain fatty acids is enhanced in X-linked adrenoleukodystrophy [published correction appears in Mol Genet Metab. 2008 Mar;93(3):350]. Mol Genet Metab. 2005;84(2):144-151. doi:10.1016/j.ymgme.2004.09.015
[2] Yska HAF, Engelen M, Bugiani M. The pathology of X-linked adrenoleukodystrophy: tissue specific changes as a clue to pathophysiology. Orphanet J Rare Dis. 2024;19(1):138. Published 2024 Mar 28. doi:10.1186/s13023-024-03105-0
[3] Jaspers YRJ, Yska HAF, Bergner CG, et al. Lipidomic biomarkers in plasma correlate with disease severity in adrenoleukodystrophy. Commun Med (Lond). 2024;4(1):175. Published 2024 Sep 10. doi:10.1038/s43856-024-00605-9
[4] Rizzo WB, Watkins PA, Phillips MW, Cranin D, Campbell B, Avigan J. Adrenoleukodystrophy: oleic acid lowers fibroblast saturated C22-26 fatty acids. Neurology. 1986;36(3):357-361. doi:10.1212/wnl.36.3.357
[5] Terluk MR, Tieu J, Sahasrabudhe SA, et al. Nervonic Acid Attenuates Accumulation of Very Long-Chain Fatty Acids and is a Potential Therapy for Adrenoleukodystrophy [published online ahead of print, 2022 Apr 4]. Neurotherapeutics. 2022;10.1007/s13311-022-01226-7. doi:10.1007/s13311-022-01226-7
[6] Figure. 1 Created with BioRender.com


Figure 1. Schematic diagram of study design