Original ContributionPlasmalogen phospholipids protect internodal myelin from oxidative damage
Graphical abstract
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.
Section snippets
Mice
Mice of the DDY inbred strain were kindly provided by Dr. T. Seyfried (Boston College Biology Department). WT and Pex7 KO littermates were obtained from heterozygous mating pairs, genotyped, and maintained as previously described [40]. Animals were sacrificed by cervical dissociation before dissection of sciatic and optic nerves, which represented the peripheral and central nervous systems, respectively. All animal procedures were conducted in accordance with protocols approved by the
Nerve myelin structure is disrupted after exposure to a ROS-generating system
To develop an in vitro ROS-generating system that induces structural alterations of myelin, we tested the ability of a copper (Cu) and hydrogen peroxide (HP)-based oxidant system (Cu/HP). This system had previously been shown to induce oxidative modification of biological molecules via site-specific hydroxyl radical production [11]. After the incubation of freshly dissected sciatic and optic nerve segments from WT DDY mice with either, neither, or both Cu and HP, their X-ray diffraction
Discussion
We have demonstrated by X-ray diffraction (XRD) that ex vivo exposure of unfixed sciatic and optic nerves to ROS-generating systems caused considerable structural changes in internodal myelin. XRD is a highly sensitive technique for quantitating the multilamellar structure of internodal myelin in whole nervous tissue—e.g., intact sciatic and optic nerves, spinal cord, and spinal roots—immediately after being dissected and without chemical fixation, or even after incubation in defined solutions
Conclusions
We have identified two ROS-generating systems capable of causing large changes in the packing of the membranes in internodal myelin of sciatic and optic nerves. Our results suggest that the Cu/OP/HP-induced compaction depended on membrane-targeted hydroxyl radical production, as evidenced by the protective roles of a hydroxyl radical scavenger and the water-soluble metal cation chelator EDTA. Significant decreases in the amounts of myelin proteins as well as increasing protein carbonylation and
Acknowledgments
This research was supported by Association Européenne contre les Leucodystrophies–ELA (ELA Foundation), Grants ELA2006-054C1, ELA2008-009C4, ELA2010-042C5), the Fondation pour l’aide à la recherche sur la Sclérose en Plaques (ARSEP Foundation), Boston College Institutional Research Funds, the Undergraduate Research Fellowship Program at Boston College, and by FEDER Funds through the Program–COMPETE, and by National Funds through FCT–Fundação para a Ciência e a Tecnologia under the projects
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2023, InnovationCitation Excerpt :Given their ether-linked alkyl chain, plasmalogens possess a hydrophobic tail with a narrower cross-sectional area, which increases liquid order in a cell membrane.72,73 Other roles attributed to plasmalogens include serving as a reservoir for arachidonic acid, modulating intracellular radical oxygen species propagation, regulating cell death, and contributing to lipid raft formation.74,75,76,77,78 Plasmalogen deficiency through knockout of peroxisome-related genes, such as Pex7 and Gnpat, is detrimental to myelination and myelin stability.79,80
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2022, BiochimieCitation Excerpt :Of particular interest are the ePE since plasmalogen content is closely associated with myelination level [9]. In addition, plasmalogens increase myelin stacking and act as endogenous antioxidants protecting other lipids from further oxidation [9,23]. Cholesterol, also a major lipid of brain and myelin, increased significantly between P15 and P40 [24].
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- 1
Current address: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637
- 2
Current address: ReNovo Neural, Inc., Cleveland, OH 44106