Plasmalogen phospholipids protect internodal myelin from oxidative damage

Highlights

• X-ray diffraction was used to probe the effects of ROS on myelin membranes.

• ROS produced extensive disruption of myelin in unfixed CNS and PNS nerves.

• Nerves from plasmalogen-deficient mice were substantially more prone to damage.

• A hydroxyl radical scavenger in the ROS solution prevented the damage.

• Plasmalogen phospholipids serve as endogenous antioxidants in myelin.

Abstract

Reactive oxygen species (ROS) are implicated in a range of degenerative conditions, including aging, neurodegenerative diseases, and neurological disorders. Myelin is a lipid-rich multilamellar sheath that facilitates rapid nerve conduction in vertebrates. Given the high energetic demands and low antioxidant capacity of the cells that elaborate the sheaths, myelin is considered intrinsically vulnerable to oxidative damage, raising the question whether additional mechanisms prevent structural damage. We characterized the structural and biochemical basis of ROS-mediated myelin damage in murine tissues from both central nervous system (CNS) and peripheral nervous system (PNS). To determine whether ROS can cause structural damage to the internodal myelin, whole sciatic and optic nerves were incubated ex vivo with a hydroxyl radical-generating system consisting of copper (Cu), hydrogen peroxide (HP), and ortho-phenanthroline (OP). Quantitative assessment of unfixed tissue by X-ray diffraction revealed irreversible compaction of myelin membrane stacking in both sciatic and optic nerves. Incubation in the presence of the hydroxyl radical scavenger sodium formate prevented this damage, implicating hydroxyl radical species. Myelin membranes are particularly enriched in plasmalogens, a class of ether-linked phospholipids proposed to have antioxidant properties. Myelin in sciatic nerve from plasmalogen-deficient (Pex7 knockout) mice was significantly more vulnerable to Cu/OP/HP-mediated ROS-induced compaction than myelin from WT mice. Our results directly support the role of plasmalogens as endogenous antioxidants providing a defense that protects ROS-vulnerable myelin.

Introduction

Incomplete mitochondrial reduction of 1–3% of our respired oxygen results in the formation of superoxide radical anions [1]. The potential toxicity of these reactive oxygen species (ROS) [2], [3] is underscored by the fact that the enzymes superoxide dismutase and catalase evolved to catalyze the reduction of superoxide to hydrogen peroxide and the dismutation of hydrogen peroxide (HP). Oxidative stress (OS), which results when ROS production exceeds the cell’s ability to scavenge them, is linked to aging [4], [5], neurodegeneration [6], Alzheimer’s disease (AD) [7], multiple sclerosis (MS) [8], and other conditions [9]. In the case of MS, the tissue pathology is characterized by an inflammatory demyelination that correlates with oxidative damage. Moreover, MS lesions exhibit an abnormal increase in carbonyl degradation products resulting from lipid acyl chain degradation [10], strongly suggesting that oxidative damage may contribute to demyelination.

The superoxide radical anion is, itself, relatively unreactive; however, it can be converted to the highly reactive hydroxyl radical, which may produce indiscriminate damage to any neighboring biomolecules [1], [11], [12]. Transition metals such as copper and iron are essential for the production of hydroxyl radicals [13], [14], [15], [16] in a conversion referred to as the metal-catalyzed Haber-Weiss reaction. This reaction occurs in two steps: first the metal species is reduced by superoxide, and then the reduced metal reacts with available hydrogen peroxide to produce a hydroxyl radical (·OH) and reoxidized metal (the Fenton reaction). Owing to its ability to initiate lipid peroxidation [17] and to cause irreversible protein modifications [18], the hydroxyl radical is considered as the ultimate cause of biological oxidative damage [11], [19]. Oxidative damage by hydroxyl radicals occurs in a site-specific manner and in close proximity to their site of generation owing to their high reactivity [11], [20], [21]. Because transition metals are redox active and required for hydroxyl radical production, the oxidative damage largely occurs at the metal binding sites in proteins [22], [23].

In the current study we investigated the effects of ROS and oxidative damage on nerve myelin. Myelin is a lipid-rich sheath that provides a high-resistance, low-capacitance, multilamellar wrapping of axons in the peripheral nervous system (PNS) and the central nervous system (CNS), allowing for faster action potential propagation via saltatory conduction. The high lipid-to-protein ratio of myelin renders the tissue inherently vulnerable to oxidative damage [24], as membrane lipids are able to sustain the propagative, free radical chain reaction, referred to as lipid peroxidation. Nerve in the PNS may be more sensitive to OS-induced demyelination than in the CNS, as suggested by the finding that markers of lipid peroxidation are elevated in PNS but not in CNS myelin following oral administration to rats of a copper accumulation-promoting compound [25]. Moreover, levels of the antioxidant glutathione are lower in the PNS than in the CNS [26]. Intermembrane adhesion of the multilamellar sheath, and therefore myelin’s compact structure, is predominantly mediated by protein zero (P0) in the PNS and by proteolipid protein (PLP) and myelin basic protein (MBP) in the CNS (reviewed in [27]). Basic amino acid residues (histidine, lysine, arginine), which constitute 10–20% of the amino acids of these myelin proteins, are among those most susceptible to modification by ·OH [28], [29], [30]. These proteins, therefore, may be particularly prone to damage and subsequent denaturation by ROS [31], resulting in disruption of membrane compaction and compromising myelin’s insulative properties [32].

Myelin is unusually rich in the class of ether-linked phospholipids known as plasmalogens, with up to 70% of ethanolamine glycerophospholipids existing as the plasmalogen form [33], [34]. Plasmalogen-deficient mammalian cells are substantially more susceptible to OS-induced death than their plasmalogen-containing counterparts, which was the first indication of a potential role for plasmalogens as membrane-protecting antioxidants [35], [36]. Subsequent studies using lipids incorporated into Triton X-100 micelles indicate that plasmalogens interrupt the free radical chain reactions of lipid peroxidation [37]. Arguing against this notion, however, is the suggestion that the products of plasmalogen oxidation may themselves be reactive, and thus promote rather than terminate the chain of oxidative processes [38].

To determine whether plasmalogen phospholipids protect myelin sheaths from oxidative damage, we developed a ROS-generating system that reproducibly induced in ex vivo myelin structural changes which were assessed by X-ray diffraction (XRD) [39]. XRD is particularly sensitive for detecting, in chemically unfixed tissue, the periodicity and hence, the structural integrity of internodal myelin. Using wild-type (WT) and Pex7 knockout (KO) mice, a mouse model for rhizomelic chondrodysplasia punctata (RCDP) that lacks plasmalogens due to impaired biosynthesis [40], [41], we found that plasmalogen-deficient myelin in sciatic nerves was highly vulnerable to damage by ROS. Our structural results together with our biochemical findings of extensive carbonylation and aggregation of proteins in ROS-damaged nerves indicate that myelin structure is highly susceptible to ROS-mediated damage and that plasmalogens serve to protect myelin from oxidative damage.