Neurochemical Research, Vol. 19, No. 8, 1994, pp. 967-974

Metabolic Turnover of Myelin Glycerophospholipids* Pierre Morell 1,3 and A n d r e a H. Ousley 2

(Accepted September 22, 1993)

The apparent half life for metabolic turnover of glycerophospholipids in the myelin sheath, as determined by measuring the rate of loss of label in a myelin glycerophospholipid following radioactive precursor injection, varies with the radioactive precursor used, age of animal, and time after injection during which metabolic turnover is studied. Experimental strategies for resolving apparent inconsistencies consequent to these variables are discussed. Illustrative data concerning turnover of phosphatidylcholine (PC) in myelin of rat brain are presented. PC of the myelin membrane exhibits heterogeneity with respect to metabolic turnover rates. There are at least two metabolic pools of PC in myelin, one with a half life of the order of days, and another with a half life of the order of weeks. To a significant extent biphasic turnover is due to differential turnover of individual molecular species (which differ in acyl chain composition). The two predominant molecular species of myelin PC turnover at very different rates (16:0, 18:1 PC turning over several times more rapidly than 18:0, 18:1 PC). Therefore, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Possible mechanisms for differential turnover of molecular species are discussed, as are other factors that may contribute to a multiphasic turnover of glycerophospholipids. KEY WORDS: Myelin metabolism; phospholipid metabolism; phosphatidylcholine; phosphatidylethanolamine.

INTRODUCTION

pulse is propagated discontinuously down a nerve fiber, with the action potential jumping from node to node. Myelin, a specialized extension of the oligodendroglial cell plasma membrane, is wrapped in spiral fashion about a nerve axon. Initially, the " p a s s i v e " electrophysiological role of myelin, coupled with the view of myelin as a very stable and ordered structure, led to the conclusion that myelin was largely metabolically inert. Indeed, it is difficult to imagine how membrane components of the compact myelin sheath would have access to the cytoplasmic machinery involved in metabolism. However, although the bulk of mature myelin has a highly ordered, multilamellar structure containing closely apposed membranes, there are also noncompacted domains of the myelin sheath, such as the lateral loops, inner and outer loops, and the longitudinal incisures. The demonstration that myelin has fluid mosaic properties similar to other membranes (9), and that over a period of time mem-

Over a century ago, Ranvier (41) first presented the view of myelin as an electrical insulator. Although his model of the physiological role of the myelin sheath and the nodes of Ranvier were in error, he correctly concluded that the high resistance and low capacitance of the myelin sheath were essential to its function. This property of myelin is fundamental to our present understanding of saltatory conduction, whereby a nerve imDept. of Biochemistry and Biophysics and Brain and Development Research Center. 2 Dept. of Physiology, University of North Carolina, Cliapel Hill N.C. 27599. 3 Address reprint requests to: Pierre Morell, Ph.D., 322 BDRC, CB#7250, University of North Carolina, Chapel Hill, N.C. 27599. * Special issue dedicated to Dr. Marjorie Lees. 967

0364-3190/94/0800 0967507.00/0 9 1994 Plenum Publishing Corporation

968 brane components can be displaced along the membrane towards regions where there is contact with cytoplasm, removed this theoretical barrier to our current understanding of myelin as a metabolically active membrane. Early Studies of Metabolic Turnover of Myelin Glycerophospholipids Some of the early long term metabolic turnover studies in nervous tissue seemed to confirm the view that myelin lipids are metabolically stable. Davison and coworkers (12-15) injected [32p]phosphate or [14C]glycerol into developing animals, and examined the turnover Of phospholipids. They suggested that a proportion of lipids in brain were metabolically inert, that radioactive lipids persisted in white matter relative to gray matter, and that the stability of lipids in whole nervous tissue contrasted with the more rapid turnover in other tissues. In each case, evidence for the metabolic stability of brain lipids was obtained, most of which was attributed to the myelin sheath. In later studies (3,11) [32P]phosphate was used to examine turnover of phospholipids in the myelin fraction alone. They concluded that all classes of myelin phospholipid turn over at the same slow rate, and that myelin membrane turns over as a metabolic unit. One method to determine metabolic turnover is to inject a radioactive precursor and, following the time of its maximal incorporation into lipids, determine the rate of loss of label. Animals are injected with a radioactive precursor, killed at various times after injection, myelin is isolated, and the amount of radioactivity present in individual lipid classes determined. Early evidence that some myelin lipids turn over at significant rates came from such studies by Smith and Eng (44). Young rats were injected with [14C]acetate and the disappearance of radioactivity was followed for the period 2 months to 2 years after injection of labeled precursor. While cerebroside, sulfatide, sphingomyelin, cholesterol, and ethanolamine plasmalogen were relatively stable (half life of 7 months to 1 year), phosphatidylinositol (PI) and phosphatidylcholine (PC) turned over at a faster rate (half life of 5 to 8 weeks). Incorporation of radioactive precursors into myelin of young developing rats is greater than that in adults. Rapid accumulation of the myelin membrane is concentrated in a defined postnatal period. In rat, the peak rate of accumulation of myelin is between 18 and 21 days of age, with a gradual decrease in the rate of accumulation thereafter (36). Therefore, when young animals are injected with radiolabeled precursors, lipids in myelin are labeled primarily due to accumulation of myelin rather than from replacement of lipids which have been metab-

Morell and Ousley olized. In older animals, myelin accumulates at a slower rate, and incorporation of label into myelin also declines. Presumably, most of the label incorporated into myelin in the older animal is due to metabolic turnover needed for maintenance of the myelin membrane. That at least a fraction of myelin phospholipids is rapidly metabolized was concluded from the demonstration that in adult rats there is considerable incorporation of [32p]phosphate into myelin (3,19). This was an unexpected finding that did not seem compatible with the data of Smith and Eng (44), which showed incorporation of radioactive acetate into myelin lipids was almost two orders of magnitude less in adult than in young animals. The discrepancy could be resolved in light of the earlier observation that in adult rats there is considerable rapid incorporation of [32P]phosphate into phosphatidylinositol (PI) and phosphatidic acid (2,12,17). With respect to labeling of phospholipids, rapid uptake of phosphate in adults involves primarily the monoesterified phosphate groups of phosphatidic acid and mono-, di- and triphosphoinositides (18,19, see Ledeen, 1992 for review), while acetate is primarily incorporated into the long chain acyl, alkyl, and alkenyl moieties. Thus, turnover of the monoesterified phosphate groups is not coupled to turnover of acyl chains or the glycerol backbone. Myelin specific aspects of metabolism of phosphatidic acid and polyphosphoinositides (18,27), and the possible involvement of phosphoinositide metabolism in second messenger signaling within myelin is an area of active investigation (30). However, this aspect of "very rapid" metabolism of myelin lipids lies outside the scope of this review. Use of Different Precursors Leads to Difference in Apparent Half Lives The apparent half life of a particular class of myelin lipid varies with the age of the animal at the time of injection, the time interval after injection that is studied, and the precursor used. This last variable accounts for much of the variation reported for turnover of myelin lipids. Abdel-Latif and Smith (1) explicitly made the point that apparent turnover of a phospholipid in (nonmyelin) subcellular fractions differed depending upon whether the labeled precursor used was glycerol, phosphate, or the base moiety. This is because some moieties, when released from degraded phospholipid, are efficiently reutilized for synthesis of phospholipids, resulting in apparent long half lives. For example, reutilization of fatty acids in whole brain has been demonstrated (45). Thus, 14C originally in precursors such as acetate or glucose is incorporated into acyl chains of phospholipids and has a long apparent half life because, follow-

Metabolic Turnover of Myelin Glycerophospholipids

969

ing partial degradation of the phospholipid, acyl chains can be reutilized. Phosphate and choline also have long half lives in phospholipids because these moieties also are reutilized. The pathways for incorporation of radioactive precursors into a glycerophospholipid by either synthesis de novo or reutilization of certain moieties are summarized (Fig. 1).

dehydrogenase which interconverts glyceroi-3-phosphate with dihydroxyacetone phosphate. This reaction results in the loss of tritium to water (4). Therefore, an assumption for the study of Miller et al. (33), was that the rate of loss of 3H label from glycerol gives the most accurate estimate of phospholipid turnover rate. A refinement in this study was that even with this precursor, with time there is metabolic randomization of label to other positions on gycerol and to acyl moieties, and correction for this must be made (33). The metabolic turnover rates of various moieties of myelin phospholipids were directly compared with that of the glycerol backbone by using a double label experimental design (Fig. 2). Phospholipids in rat brain were labeled in vivo by intracranial injection of [2-3H]glycerol, simultaneously with one other radiolabeled precursor, at an age when myelin was being deposited at a rapid rate. The second precursor was either [14C]acetate to label acyl and alkenyl moieties, [32p]phosphate to label the phosphoryl moiety, or [14C]choline or [14C]ethanolamine to label the base moiety (data in Fig. 2, 3 is with respect to labeled choline). Myelin was isolated at various times after injection (2 to 80 days), individual classes of phospholipid were separated by two dimensional thin layer chromatography, and the 3H/32p or 3H/14C ratio was determined for each phospholipid class. Some of the results of this study are reviewed with respect to turnover of phosphatidylcholine (PC). The decay of [2-3H]glycerol label in PC was plotted (Fig. 3). A half life of 25 days was calculated for turnover between 15 and 80 days (a discussion of two phases of turnover is found later). In the inset, the ~H/14C ratio in myelin PC at various times following injection of [23H]glycerol and [14C]choline is shown. While it is intuitively obvious that the glycerol moiety is turning over more rapidly than choline, this can be quantitated if the 3H/14C ratio is divided into the absolute [3H]glycerol radioactivity. For the period between 15 and 80 days after injection, choline has a turnover rate of 39 days vs. 25 days for glycerol. Analogous experiments were performed with other radioactive precursors. Of interest is that the half life was 30 days with [32p]phosphate and 54 days with [~4C]acetate. While data is only shown here for myelin PC, a similar analysis was performed for phosphatidylethanolamine (PE) of myelin. For the period between 15 and 80 days, the half life was 25 days with [2-3H]glycerol, 33 days with [14C]ethanolamine, 30 days with [32P]phosphate, and 125 days with [*4C]acetate (vs. 54 days for PC). Therefore, one conclusion from this study was that white the glycerol, base, and phosphate moieties turn over at about the same rate in PC and PE, the acyl chains are more extensively reutilized

A comprehensive study of the relationship between radioactive precursor used and apparent half life for turnover of individual classes of myelin lipids was carried out by this laboratory (33). This study took advantage of the fact that [2-all]glycerol is a good precursor for pulse labeling the glycerol backbone of brain lipids since it is subject to minimal reutilization. Label in glycerol3-phosphate released from degraded phospholipid is rapidly lost because of the activity of glycerol phosphate

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MoreU and Ousley

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20 40 60 80 TIME AFTER INJECTION(days) Fig. 3. Loss of radioactivity in PC of myelin following injection of [2-3H]glycerol into young rats. The loss of label in myelin PC following injection of [2-3H]glycerolis mulfiphasic. The half-life of the rapid and slow phases, and the percentage of phosphotipid in each pool, can be calculated (see text for discussion). Inset shows the decline in 3H/ t4C ratio in myelin PC after simultaneous injection of [2-3H]glycerol and [~4C]choline; this indicates that choline is reutilized.

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data, although the absolute turnover time may be somewhat in error, conclusions such as a difference between PC and PE with respect to turnover of gIycerol vs. acyl moieties can be established with confidence.

[~H]~ J L - - J TIME Fig. 2. Experimental Designs for study of glycerophospholipid turnover. In one design, rats were injected with [2-3H] glycerol, and another labeled precursor (data with [~4C]cholineis illustrated). At several subsequent times animals were killed, brains removed, and myelin isolated. Lipids were extracted and lipid classes separated from each other by two dimensional thin layer chromatography. For each lipid class loss of radioactivity with time, and change in isotope ratio are plotted. From these can be derived turnover times of the lipid class, and turnover of the two labeled moieties relative to each other (33). Considerable further information can be gained by analysis of individual molecular species within each phospholipid class. Each phospholipid class (a spot on the chromatogram) is eluted and hydrolyzed to diacylglycerides. Following dinitrobenzoylation, the individual diacytglycerol species can be separated from each other by high performance thin layer chromatography(HPLC). With time, increase in percent representation of label in one diglyceride species suggests greater metabolic stability of that species, relative to other species.

for the synthesis of PE than for PC. The double label design for determination of relative turnover of moieties such as glycerol and choline, is less subject to the high injection variability typical in these types of experiments than is comparison of data from independent sets of animats (33, for discussion). Thus, in reference to the above

Each Phospholipid Class Has More Than One Rate of Turnover It is evident from the decay curve in Fig. 3 that even for an individual class of myelin phospholipid such as PC, metabolic turnover as determined with the use of radioactive glycerol is multiphasic. This had previously been noted for total brain phospholipids (25,26). It is generally accepted that there are " f a s t " and " s l o w " pools within individual myelin phospholipid classes, therefore, the time course of decay is often arbitrarily divided into rapid and slow phases. If the decay curve (Fig. 3) is approximated as having two phases, it can be resolved into two components with very different apparent turnover rates. A half life of 25 days is calculated for turnover between 15 and 80 days. The amount of radioactivity in this " s l o w l y " turning over pool is subtracted from the total radioactivity to obtain a corrected half life of 6 days for the fast phase of turnover (Figure 3, see 25 for procedure). The proportion of lipid in each pool can be obtained from the following equation (25). % slow pool = (R~ x ts)/[(R~ x t~) + (R r x tr)] where,

Metabolic Turnover of Myelin Glycerophospholipids

971

R = the proportion of total radioactivity in the sIow or rapid pool at the time of maximal radioactivity t = the half life of the slow or rapid pool.

faster rate shouId decrease with time, relative to that in species that turn over at a slower rate.

By this calcuIation, 16% of the myelin PC is in the fast pool with the remainder in the slow pool. Similar multiphasic metabolic turnover was also demonstrated with other precursors (33).

Multiphasic Turnover May Be, in Part, Accounted for By Different Molecular Species Turning Over at Different Rates Much of the literature concerning turnover of myelin lipids carries the implicit assumption that each class of lipid, such as PC or PE, is metabolically homogeneous. It has been shown that this assumption does not hold for sphingomyelin. The two predominant molecular species of sphingomyelin, those with stearic and lignoceric acid in the N-acyl position, turn over at different rates (20). The situation with glycerophospholipids is potentially more complicated because various combination of fatty acids at the sn-1 and sn-2 positions lead to great compositional heterogeneity. While it is now generally accepted that there are within a class of myelin phospholipid fast and slow pools, the relationship of this to turnover of individual molecular species has only recently been investigated (38). In that study (experimental design in Fig. 2), phospholipids of rat brain were labeled with [2-3H]glycerol. Myelin was isolated at 1 and 15 or 30 days after injection, phospholipid classes were separated by two dimensional thin layer chromatography and hydrolyzed to diacylglycerols. The diacylglycerols from each lipid class were derivatized with 3, 5-dinitrobenzoyl chloride, the individual molecular species resolved by reverse phase HPLC, and the distribution of radioactivity among different molecular species determined. Since precursor was administered during the period of maximal myelin deposition, phospholipid in both the "fast" and " s l o w " pools of myelin phospholipid were labeled. Both pools are represented in the individual diacylglycerol species resolved by HPLC at 1 day post injection. By 15 or 30 days post injection, much of the label in the "fast" pool has disappeared; therefore, the diacylglycerol species remaining represent predominantly the " s l o w " pool. If individual molecular species of myelin PC turn over at the same rate, then one would expect to see no change in the distribution of ~H-radioactivity among individual molecular species with time following precursor injection. Conversely, if molecular species turn over at different rates, the percentage of total label in the molecular species that turn over at a

The results of the above described experimental approach are itiustrated with respect to the distribution of radioactivity among individual molecular species of myelin PC (Fig. 4). Between 1 and 30 days the fraction of label in palmitoyl, oleoyl (16:0, 18:1) PC decreases while that in stearoyl, oleoyI (18:0, 18:1) PC increases. This indicates that 16:0, 18:1 PC turns over more rapidly than 18:0, 18:1 PC. If we assume that the most stable species of myelin PC (18:0, 18:1 PC) was turned over with a half life of 25 days (the value reported for the slow phase of myelin PC turnover, 33), then the half life for the other molecular species can be calculated (see 38). By that calculation, 16:0-18:1 PC turns over between 1 and 15 days post-injection with a half life of 6 days. Therefore, differential turnover of the two predominantly labeled molecular species, 16:0, 18:1 and 18:0, 18:1 PC, may account for much of the "biphasic" turnover originally observed for myelin PC (33).

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972 Complications Concerning Above Interpretation: Structural Features May Influence Metabolic Turnover

Phospholipid Turnover within Different Myelin Subfractions. As noted earlier, while the bulk of mature myelin has a compact, multilamellar structure, there are noncompacted regions of the myelin membrane such as the lateral loops, inner and outer loops, and longitudinal incisures. These are lysed during myelin preparation so that cytoplasmic contents can be separated from membranous material by sedimentation. In addition to these myelin domains, the membrane fraction contains also a transition region containing the most recently deposited myelin; this material also may not be fully compacted. Thus, purified myelin can be subdivided into myelin subfractions, which differ in particle size and density, and which can be separated by centrifugation on discontinuous sucrose gradients. The lighter subfractions are enriched in large, multilamellar whorls of membrane that resemble, and are almost certainly derived from, compact myelin. The dense subfraction is composed of vesicles, small membrane fragments, and occasional large myelin fragments (5,21,31,49). It is assumed that different subfractions, which differ in morphology and composition, originate from different domains of the myelin sheath (reviewed in 35). These myelin subfractions have metabolic significance; at least some lipid and protein components of myelin are involved in precursor-product relationships in that they first appear in the dense myelin subfraction and only subsequently in the lighter, presumably more mature and compacted, myelin (5-8 for review). The question arises as to whether the "subfractions" correspond to compartments of glycerophospholipids which turn over at different rates. This can not easily be tested directly because of the above mentioned time dependent transfer of material from denser to lighter subfractions. However, a double label experimental design was applied to analysis of myelin subfractions (38). Rats were labeled in vivo by injection of [14C]glycerol at 20 days of age, followed by injection of [2-3H]glycerol 33 days later, and animals were sacrificed at 1 day after the 3H injection. Myelin subfractions were prepared and the 3H/14C ratio in PC of myelin subfractions determined. While both the "fast" and "slow" pools are labeled with 3H at 1 day after injection, by this time, much of the 14C administered earlier and incorporated into the "fast" pool will have disappeared. Therefore, a high 3H/~4Cratio in a particular myelin subfraction should reflect rapid turnover in that membrane fraction relative to that in other subfractions. The results indicated that there was indeed some

Morell and Ousley

heterogeneity in turnover of lipids in the different subfractions (38), the denser subfraction with relatively greater metabolic turnover. Because the densest subfraction contains only a small percentage of the total myelin fraction, we assume that the apparently rapid turnover is due to transfer of material to lighter subfractions (maturation of myelin). The results of this experiment also demonstrate that the faster turnover of (16:0, 18:0) PC, relative to that of (18:0, 18:1) PC, was not compartmentalized within any of the major myelin subfractions (38). Asymmetry of Myelin Membrane. One explanation which has been offered for multiphasic turnover is based on the assumption that newly-synthesized lipid is incorporated into both the external and cytoplasmic faces of the membrane during membrane biogenesis. Lipids localized to the external face, which are presumably less accessible to the cytosolic compartment, may be the source of the more metabolically stable pool (25,34). Relevant is a calculation from the data of Miller et al. (33 - obtained by the analysis described in a preceding section) indicating that only 16% of PC, but 38% of PE, is in the "rapid" turnover pool. This may relate to the generally differentially asymmetric disposition of phospholipids in membranes with PC preferentially in the extracellular face and PE preferentially in the cytoplasmic face. Data on these distributions for myelin is not available, except for the preferential localization of phosphatidylethanolamine (plasmalogen) on the cytoplasmic face (29). 9"New'"myelin Forms a Compartment. Another relevant factor relates to the observation that, when the turnover of myelin PC is followed after injection of [23H]glycerol in adult rats (34), the rapidly turning over lipids are preferentially labeled. Possibly newly-synthesized lipids are incorporated only during membrane biogenesis (during the developmental period when myelin accumulates rapidly) into a compartment of the myelin sheath where lipids turn over slowly. In this interpretation, in young animals lipid incorporated into myelin is quickly buried beneath the "newer" outermost lamellae of the rapidly accumulating myelin membrane. Lipids in the outermost layers may be more accessible to catabolizing enzymes or lipid exchange proteins (23,34). Possible Mechanisms for Differential Turnover of Different Molecular Species of Glycerophospholipids

Lipase Specificity and Remodeling. Phospholipid degradation in mammalian cells involves deacylation catalyzed by phospholipase A1 or A2, followed by action of a lysophospholipase of the appropriate specificity.

Metabolic Turnover of Myelin Glycerophospholipids Preferential catabolism of certain molecular species may reflect in vivo substrate specificities of the phospholipase and lysophospholipase enzymes involved in phospholipid degradation. Proposed models, based on crystal structures for the phospholipase A 2 enzymes, suggest that the enzyme interacts partially with the acyl chains (43,47,46). Differential specificity of these enzymes could also be involved in remodeling (change of the acyl chain composition of a phospholipid via deacylation and reacylation reactions, with retention of the glycerol backbone). In rat liver, rapid turnover of certain molecular species of PC involves remodeling (reviewed in 24, 32) rather than complete catabolism of these species. However, it has been shown in rat hepatocytes that 16:0, 22:6 PE is catabolized at a faster rate than 18:0, 22:6 PE or other molecular species (42). While the oligodendroglial cell body is the primary site for biosynthesis and catabolism of lipids in myelin, many enzyme activities related to phospholipid synthesis (or remodeling) and catabolism are intrinsic to myelin (recently reviewed, 30), and thus this process might be carried out even in compacted myelin. Involvement of Phospholipid Transfer Protein. Another possibility is that lipid transfer proteins, found ubiquitously in the cytosolic fraction of eukaryotic cells (reviewed in 48), are involved in the retrieval of phospholipids from cellular membranes prior to their degradation. While it has previously been suggested that transfer proteins may play a role in membrane biogenesis (16), the physiological role of these proteins remains obscure. Transfer proteins could mediate both the movement of newly-synthesized phospholipids from the endoplasmic reticulum to other subcellular membranes and subsequent retrieval from those membranes. In this context, it should be noted that a role for PC transfer protein has been suggested in recycling of pulmonary surfactant (which has a high 16:0, 16~0 PC content) between the alveolar surface and lamellar bodies of lung. In vitro, PC transfer protein from lung transfers 16:0, 16:0 PC at a rate 1.5-fold higher than 18:1, 18:1 or 16:0, 20:4 PC (22). PC transfer protein (and PI transfer protein) have distinct binding sites for the sn-1 and sn-2 acyl chains. In one study, the binding affinity of PC transfer protein from bovine liver for different molecular species of PC relative to that of a PC analog was determined (28). The binding affinity of PC transfer protein for molecular species containing 16:0 in the sn-1 position is higher than that for those containing 18:0 at the sn-1 position, e.g. the relative affinity for 16:0, 18:1 PC is 2.5-fold higher than for 18:0, 18:1 PC. This matches quite well with the differences observed in half-life for myelin PC of precisely these acyl chain compositions.

973 SUMMARY Glycerophospholipids in myelin, although more metabolically stable than in most other membranes, are subject to continuous degradation and replacement. In addition, the metabolic turnover of a glycerophospholipid class in myelin is multiphasic, e.g. PC and PE in myelin from rat brain exhibit more than one turnover rate. Mechanisms specific to the unique structure of myelin may contribute to compartmentalization of two or more metabolic pools. Our data, however, indicate that differential turnover of individual molecular species accounts for much of the multiphasic turnover observed for glycerophospholipids in myelin. This may have general significance with respect to membrane lipid turnover as multiphasic turnover of glycerophospholipid classes has been observed in neoplastic mast ceils (40), MOPC 41 myeloma cells (10), microsomal membranes from rat liver (37), and various subcellular fractions from brain (39).

ACKNOWLEDGMENT Supported by USPHS Grant NSl1615.

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