(T1530-05-29) Nervonic Acid - A Potential Therapeutic Targeting Mitochondrial Dysfunction and Cellular Oxidative Stress

Purpose: Adrenoleukodystrophy (ALD) is an X‐linked rare genetic condition characterized by the accumulation of saturated very long-chain fatty acids (VLCFA), mainly hexacosanoic acid (C26:0) due to reduced β-oxidation of VLCFAs in peroxisomes.1 Vast phenotypic variability is observed in this disease, of which Adrenomyeloneuropathy (AMN) which commonly starts in adulthood and affects the spinal cord and nerves, is the most frequent manifestation.2 However, oxidative lesions were observed prior to the neurological symptoms in the ALD mouse model, which recapitulates the AMN features. In addition, redox disequilibrium including an increased level of reactive oxygen species (ROS) and diminished mitochondrial inner membrane potential was observed in human ALD fibroblasts.3 Cumulative evidence indicates that C26:0-induced oxidative stress is the leading factor in disease progression, which is considered to be a promising therapeutic target for ALD. Monounsaturated fatty acids were shown to reduce VLCFA accumulation in fibroblasts derived from patients and healthy controls, thus indicating that dietary fatty acids can be used to lower the toxic C26:0 levels in patients with ALD and delay disease onset or progression.4,5 Considering the biochemical benefits of nervonic acid (NA, C24:1), a monounsaturated fatty acid that naturally exists in human milk, we try to explore it as a potential therapeutic for ALD. We had previously shown that NA can significantly lower C26:0 levels in fibroblasts.4 To further decipher the effects of NA on mitochondrial function and cellular oxidative stress, a series of assays were conducted using AMN patient-derived fibroblasts treated with increasing concentrations of NA.

Methods: Human dermal fibroblasts (GM17819) were seeded on chamber slides with a density of 12,500 cells per well and incubated at 37˚C. At ~80% confluence, cells were treated with ethanol (vehicle, < 0.1%) and NA (5, 20, and 50 µM) for 5 days. Untreated AMN fibroblasts were considered as the control group. After 5 days of incubation, cells were stained with various fluorogenic reagents for different purposes: MitoSOX™ Red (5 µM) was used to visualize mitochondrial superoxide in live cells; CellROX (5 µM) Green was used to detect and quantify ROS in live cells; MitoTracker™ Green (200 nM) was used to localize mitochondria in live cells, and 4',6-diamidino-2-phenylindole (DAPI), a DNA binding dye (Sigma-Aldrich, MO, US). Cell localization and fluorescence visualization were then completed using All-in-One Fluorescence Microscope BZ-X810 (Keyence, Osaka, Japan). Additionally, Seahorse XF technology (Agilent, CA, US) was adopted using the Cell Mito Stress Test assay to measure mitochondrial respiration and ATP production in cells with or without NA treatment, thus confirming the benefit of NA in improving mitochondrial function.

Results: Our results showed that both mitochondrial superoxides and overall ROS within cells were significantly reduced by 20 µM and 50 µM NA treatment, suggesting its antioxidant activity and maintenance of ROS homeostasis. The mitochondria localized by MitoTracker™ were significantly increased at 20 µM and 50 µM of NA treatment, demonstrating its ability in improving mitochondrial content. These observations were further confirmed by the Seahorse assay, which showed treatment with 5 µM and 20 µM of NA for 24 hours increased the mitochondrial function and ATP production.

Conclusion: As a monounsaturated fatty acid, NA seems to be a very promising candidate for attenuating cellular oxidative stress and restoring mitochondrial dysfunction. It appears that cells treated with 20 µM NA have the best effect on all the target measures. The underlying mechanisms would include direct interaction with VLCFA to reduce its abnormal accumulation and facilitate ATP production within mitochondria. Overall, investigation of the effect of NA in VLCFA-induced cell dysfunction will not only benefit ALD patients but also patients suffering from other neurological diseases. Further studies to understand exposure-response relationships to identify optimal dosage regimens are crucial for successful translation to preclinical models and humans.

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] Fourcade S, Ferrer I, Pujol A. Oxidative stress, mitochondrial and proteostasis malfunction in adrenoleukodystrophy: A paradigm for axonal degeneration. Free Radic Biol Med. 2015;88(Pt A):18-29. doi:10.1016/j.freeradbiomed.2015.05.041
[3] Fourcade S, López-Erauskin J, Galino J, et al. Early oxidative damage underlying neurodegeneration in X-adrenoleukodystrophy. Hum Mol Genet. 2008;17(12):1762-1773. doi:10.1093/hmg/ddn085
[4] 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
[5] 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

Figure 1. Schematic diagram illustrating cellular dysfunction in Adrenoleukodystrophy (ALD) cells and the effect of Nervonic acid (NA) on the pathology (Created with BioRender.com)



Figure 2. Nervonic acid improves mitochondrial and cellular oxidative stress in Adrenoleukodystrophy (ALD) cells. Bar plots indicating quantification of fluorescence intensity of (A) MitoSOX™, (B) CellROX, and (C) MitoTracker™ in ALD cells after 5 days of NA treatment. Data is normalized by the control which is set to 100% fluorescence. Data from all groups are from 3 independent experiments performed in duplicate and shown as mean ± SEM. One-way Analysis of Variance (ANOVA) followed by Dunnett's test was used to compare control vs. vehicle and treatment groups (* p<0.05, ** p<0.01, *** p<0.001 were statistically significant). # p<0.01 denotes a significant difference between NA 5 µM and NA 50 µM groups based on an unpaired Student's t-test.