Naturally Occurring Nervonic Acid Ester Improves Myelin Synthesis by Human Oligodendrocytes

Abstract

The dysfunction of oligodendrocytes (OLs) is regarded as one of the major causes of inefficient remyelination in multiple sclerosis, resulting gradually in disease progression. Oligodendrocytes are derived from oligodendrocyte progenitor cells (OPCs), which populate the adult central nervous system, but their physiological capability to myelin synthesis is limited. The low intake of essential lipids for sphingomyelin synthesis in the human diet may account for increased demyelination and the reduced efficiency of the remyelination process. In our study on lipid profiling in an experimental autoimmune encephalomyelitis brain, we revealed that during acute inflammation, nervonic acid synthesis is silenced, which is the effect of shifting the lipid metabolism pathway of common substrates into proinflammatory arachidonic acid production. In the experiments on the human model of maturating oligodendrocyte precursor cells (hOPCs) in vitro, we demonstrated that fish oil mixture (FOM) affected the function of hOPCs, resulting in the improved synthesis of myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein, as well as sphingomyelin. Additionally, FOM reduces proinflammatory cytokines and chemokines, and enhances fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) synthesis by hOPCs was also demonstrated. Based on these observations, we propose that the intake of FOM rich in the nervonic acid ester may improve OL function, affecting OPC maturation and limiting inflammation.

Keywords: fish oil; nervonic acid; oligodendrocytes; remyelination.

Figure 1

Human oligodendrocyte precursor cells (hOPCs) incorporate and metabolize nervonic acid. (A) GC/MS lipid profiling of the whole brain tissue revealed that nervonic acid (NA) ester (red arrows) is not synthetized during EAE in contrast to arachidonic acid ester (blue arrows) known for its proinflammatory properties. (B) Human phorbol 12-myristate 13-acetate (PMA)-stimulated MO3.13 cells, as the cellular model of oligodendrocyte progenitor cells (OPC) polarization to mature myelin-producing oligodendrocytes, were used to determine an effect of fish oil mixture (FOM) supplementation on OPC function. LO was used as the negative control. PMA-stimulated MO3.13 cells were incubated with 5% FOM or 5% LO for 72 h. (C middle right panel) Sphingomyelin consists of a phosphocholine head group, sphingosine, and fatty acid esters which are bound via an amide bound to a sphingosine base [31,34]. Fatty acid esters were detected by the GC/MS method. (C) NA, opposite to palmitic and stearic acid is synthetized de novo by maturating OPCs. Palmitic and stearic acid esters are constitutively presented in hOPCs, and can be directly used for sphingomyelin synthesis or can also be used as substrates for NA synthesis with cis-13-dicosenoic acid as an intermediate product (blue arrows). During FOM supplementation, NA is directly incorporated by hOPCs, while the cis-13-dicosenoic acid-dependent pathway is inhibited (red arrow). The rates of acid esters were measured at three time points (24 h, 48 h, and 72 h) in lyophilisates of hOPCs incubated with FOM or LO or in medium. Data are presented as means ± SD from four independent experiments.

Figure 2

Supplementation with FOM opposite to LO during hOPC maturation enhances myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), and myelin basic protein (MBP) synthesis by mature oligodendrocytes (OLs). Immunocytochemical (ICC) characterization of hOPCs supplemented with FOM during their differentiation into mature MBP/PLP/MOG-producing OLs. Confocal z-stack analysis confirmed that hOPCs supplemented with FOM were characterized by MOG/PLP accumulation in the intracellular space during their maturation. The data from the quantitative fluorescence signal of protein analysis are presented as average area (Avg Area) from four independent experiments.

Figure 3

(A) Quantitative analysis of myelin protein mRNA expression confirmed that hOPCs supplemented with FOM contrary to LO were characterized by increased MOG, PLP, and MBP synthesis during maturation into OLs. (B) Increased global mRNA levels in hOPCs supplemented with FOM. Data are presented as means ± SD from four independent experiments.

Figure 4

Fish oil mixture prolongates OL lifespan. (A) FOM supplementation inhibited spontaneous apoptosis of OLs. Two regions, R1 and R2, were gated based on the morphology (FSC vs. SSC) of MO3.13 cells and analyzed by flow cytometry. The rates of cells undergoing a late phase of apoptosis (double-positivity for propidium iodine and Anexin V) are presented as means ± SD from four independent experiments. (B) FOM supplementation had no effect on OL lysis, as LDH concentrations in supernatants were not statistically significant compared to hOPCs cultured alone (left panel). Data (means ± SD from four independent experiments) are presented as LDH concentrations and changes were normalized to positive and negative controls (right panel) according to the formula presented in the upper panel (percentage of lysed oligodendrocytes). (C) Differential contrast microscopy (DIC) microscopy analysis presented that FOM, contrary to LO, had no effect on hOPC morphology. Red arrows indicate large lamellipodia and filipodia forming a branched network, confirming the typical structure of OLs in the presence of FOM.