Localization of Phospholipid Synthesis to Schwann Cells and Axons

Quantitative electron microscopic autoradiography was used to detect and characterize endoneurial sites of lipid synthesis in mouse sciatic nerve. Six tritiated phospholipid precursors (choline, serine, methionine, inositol, glycerol, and ethanolamine) and a protein precursor (proline) were individually injected into exposed nerves and after 2 h the mice were perfused with buffered aldehyde. The labeled segments of nerve were prepared for autoradiography with procedures that selectively remove nonincorporated precursors and other aqueous metabolites, while preserving nerve lipids (and proteins). At both the light and electron microscope levels, the major site of phospholipid and protein synthesis was the crescent-shaped perinuclear cytoplasm of myelinating Schwann cells. Other internodal Schwann cell cytoplasm, including that in surface channels, Schmidt-Lanterman incisures, and paranodal regions, was less well labeled than the perinuclear region. Newly formed proteins were selectively located in the Schwann cell nucleus. Lipid and protein formation was also detected in unmyelinated fiber bundles and in endoneurial and perineurial cells. Tritiated inositol was selectively incorporated into phospholipids in both myelinated axons and unmyelinated fibers. Like inositol, glycerol incorporation appeared particularly active in unmyelinated fibers. Quantitative autoradiographic analyses substantiated the following points: myelinating Schwann cells dominate phospholipid and protein synthesis, myelinated axons selectively incorporate tritiated inositol, phospholipid precursors label myelin sheaths and myelinated axons better than proline.     

Ether lipid metabolism. Incorporation of O-hexadecyl ethanediol into rat brain lipids.

1-O-[1'-14C]Hexadecyl ethanediol was administered intracerebrally to myelinating rat brain, and incorporation of radioactivity into brain lipids was followed over a 48-h period: (1) O-Hexadecyl ethanediol was metabolized primarily through oxidative ether bond cleavage, and much of the label was recovered in phospholipid acyl groups. (2) Substantial amounts of radioactivity were also found in choline and ethanolamine phospholipids having an O-hexadecyloxyethyl glycerol backbone. This means that alkyl ethanediol was used in glycerol ether biosynthesis as are long-chain primary alcohols. (3) Acidic hydrolysis of the ethanolamine glycerophosphatide fraction yielded also labeled hexadecanol which may indicate desaturation of 1-O-hexadecyloxyethyl 2-acyl glycerophosphoryl ethanolamine to the plasmalogen analogue. (4) Small amounts of the substrate were oxidized to O-hexadecyl glycolic acid and incorporated into the phospholipids. The substrate did not serve as precursor of O-hexadecyl ethanediol phosphorylcholine or phosphorylethanolamine in the brain.     

Composition of myelin from peripheral and central nervous systems of the squirrel monkey

Myelin was prepared from the brachial plexus and cervical spinal cord of adult squirrel monkeys (Saimiri sciureus). Brachial plexus myelin contained a larger amount of sphingomyelin and smaller amounts of cholesterol, lipid galactose, ethanolamine phosphoglyceride, choline phosphoglyceride, and alk-1-enyl ether than spinal cord myelin when compared as ratios to total lipid phosphorus. The peripheral nervous system myelin had a higher proportion of protein. All of these differences were statistically significant. Thus peripheral nervous system myelin and central nervous system myelin differ in protein content and lipid composition in this subhuman primate.     

Formation of 1-O-2'-hydroxyalkyl glycerophosphatides from 1,2-heptadecanediol in myelinating brain

1,2-Heptadecanediol-2-(14)C was administered intracerebrally to 18-day-old rats, and its incorporation, after 8 hr, into the individual aliphatic moieties of the ethanolamine glycerophosphatides was determined. Much of the radioactivity was found in a lipid fraction identified as 1-O-2'-hydroxyheptadecyl glycerol. Evidence is presented that a major portion of the precursor was incorporated into 1-O-2'-hydroxyheptadecyl-2-acyl ethanolamine phosphatides. Some of the diol administered was degraded to palmitic acid. The palmitic acid-1-(14)C derived from 1,2-heptadecanediol-2-(14)C apparently served as precursor for stearic and oleic acids, which were found as acyl groups, and for the biosynthesis of the corresponding O-alkyl and O-alk-1-enyl glycerols. The data presented prove that biological dehydration of 1-O-2'-hydroxyalkyl glycerophosphatides to the corresponding plasmalogens does not occur in myelinating brain.     

Regulation of phospholipid metabolism in differentiating cells from rat brain cerebral hemispheres in culture. Patterns of acetylcholine phosphocholine, and choline phosphoglycerides labeling from (methyl-14C)choline.

The uptake and metabolism of [14C]choline in dissociated rat brain embryo cell cultures was examined as a function of the extracellular choline concentration. Choline uptake did not follow normal Michaelis-Menten kinetics, but rather exhibited two components with apparent Km of 0.016 mM and 0.96 mM. At low choline concentrations (high affinity uptake) most of the [14C]choline label was present in the phosphocholine fraction prior to the appearance of label in phospholipids. At high choline concentrations (low affinity uptake) a large proportion of the radioactivity was converted into acetylcholine. The dissimilarities between the formation of phosphocholine and acetylcholine as a function of choline concentration might be explained by the existence of two mutually independent enzymatic activities with different Km affinities for choline. Kinetic data augmented by double label studies, suggested that formation of choline phosphoglyceride proceeds entirely via a phosphocholine intermediate. Nearly all radioactivity in the lipid fraction is incorporated into choline phosphoglycerides. A higher turnover rate of choline incorporation into choline phosphoglycerides, accompanied by an increase in the levels of glycerophosphocholine, was observed in older cultures as compared to younger cultures. The metabolic implications of these findings in cultured brain cells in comparison with other in vitro systems are discussed.     

A regional survey of myelin development: some compositional and metabolic aspects

A survey of differences in composition and metabolism of myelin from five areas of the central nervous system was made in brain and spinal cord slices of the rat from 20 days to 20 months postnatal age. Purified myelin from the forebrain areas showed a composition characteristic of immaturity longer than did myelin from the hindbrain and spinal cord. The trend of chemical maturity is in agreement with the anatomical observations that myelination begins in the hindbrain and proceeds rostrally. Myelin recovery per 100-mg slice increased continually from 20 days to 20 months of age, while the uptake of [1-(14)C]acetate into myelin lipid and of [1-(14)C]leucine into myelin protein decreased precipitously with age. Taking into account the continuous increase in myelin during maturation, a calculation was made of the total amount of incorporation of labeled material into lipids or proteins per 100-mg slice for each region at each age. The metabolic characteristics of myelin from the cerebral cortex (including the corpus callosum), the thalamic area, and the cerebellum were very similar, while myelin from brainstem and spinal cord was metabolically more active, especially at the early ages. Synthesis of lipid in the myelin sheath represents about 50% of the lipid synthesis of the whole brain and about 75% of that of the spinal cord. The proportion of myelin-related protein synthesis is much less, probably less than 10% of the protein synthesis occurring in whole brain and about 15% of that in the spinal cord except at early ages.     

Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with 14C-dihomo-gamma-linolenic acid, 14C-arachidonic acid or 14C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.     

Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).     

Differentiation-arrested rat fetal lung in primary monolayer cell culture. IV. Paradoxical effect of a "fetal" pO2 on precursor incorporation into phospholipids and hormone responsiveness

Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages. In contrast to previously published observations with cultures in a pO2 of approximately 142 mm Hg, cultures developed in a pO2 of approximately 30 mm Hg, close to the normal fetal arterial pO2, have improved plating efficiency and a slightly increased growth rate. They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin. The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO2 approximately 30 mm Hg, despite preservation of an immature morphology. Electron microscope observations revealed no gross differences between immature cultures developed at either pO2. The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung.     

Phosphate groups modifying myelin basic proteins are metabolically labile; methyl groups are stable

Young and adult rats received intracranial injections of [33P]orthophosphoric acid. The time course of the appearance and decay of the radioactive label on basic proteins in isolated myelin was followed for 1 mo. Incorporation was maximal by 1 h, followed by a decay phase with a half-life of approximately 2 wk. However, radioactivity in the acid-soluble precursor pool (which always constituted at least half of the total radioactivity) decayed with a similar half-life, suggesting that the true turnover time of basic protein phosphates might be masked by continued exchange with a long-lived radioactive precursor pool. Calculations based on the rate of incorporation were made to more closely determine the true turnover time; it was found that most of the phosphate groups of basic protein turned over in a matter of minutes. Incorporation was independent of the rate of myelin synthesis but was proportional to the amount of myelin present. Experiments in which myelin was subfractionated to yield fractions differing in degree of compaction suggested that even the basic protein phosphate groups of primarily compacted myelin participated in this rapid exchange. Similar studies were carried out on the metabolism of radioactive amino acids incorporated into the peptide backbone of myelin basic proteins. The metabolism of the methyl groups of methylarginines also was monitored using [methyl-3H]methionine as a precursor. In contrast to the basic protein phosphate groups, both the peptide backbone and the modifying methyl groups had a metabolic half-life of months, which cannot be accounted for by reutilization from a pool of soluble precursor. The demonstration that the phosphate groups of myelin basic protein turn over rapidly suggests that, in contrast to the static morphological picture, basic proteins may be readily accessible to cytoplasm in vivo.     

A novel fluorescent probe that is brain permeable and selectively binds to myelin.

Myelin is a multilayered glial cell membrane that forms segmented sheaths around large-caliber axons of both the central nervous system (CNS) and peripheral nervous system (PNS). Myelin covering insures rapid and efficient transmission of nerve impulses. Direct visual assessment of local changes of myelin content in vivo could greatly facilitate diagnosis and therapeutic treatments of myelin-related diseases. Current histologic probes for the visualization of myelin are based on antibodies or charged histochemical reagents that do not enter the brain. We have developed a series of chemical compounds including (E,E)-1,4-bis(4'-aminostyryl)-2-dimethoxy-benzene termed BDB and the subject of this report, which readily penetrates the blood-brain barrier and selectively binds to the myelin sheath in brain. BDB selectively stains intact myelinated regions in wild-type mouse brain, which allows for delineation of cuprizone-induced demyelinating lesions in mouse brain. BDB can be injected IV into the brain and selectively detect demyelinating lesions in cuprizone-treated mice in situ. These studies justified further investigation of BDB as a potential myelin-imaging probe to monitor myelin pathology in vivo.     

Veratridine-Induced Depolarization Reduces the Palmitoylation of Brain and Myelin Glycerolipids

Abstract: In this study, we have investigated the effect of neuronal depolarization on the palmitoylation of myelin lipids. For this purpose, brain slices from 60-day-old rats were incubated with [³H]palmitate for 1 h in the presence or absence of various drugs. Veratridine (100 µM) reduced the incorporation of [³H]palmitate into all brain glycerolipids by 40–50%, whereas the labeling of sphingolipids was unaffected. Similar results were obtained by using [³H]glycerol as a precursor, demonstrating that veratridine also causes a reduction in the de novo synthesis of glycerolipids. Both tetrodotoxin (1 µM) and ouabain (1 mM) prevented the effect of veratridine, indicating that it is mediated through the opening of voltage-gated sodium channels and involves the stimulation of the Na⁺/K⁺ pump. Decreased levels of both ATP, due to activation of the Na⁺,K⁺-ATPase, and the precursor palmitoyl-CoA were found in the veratridine-treated slices, thus explaining the reduction in lipid synthesis. Neuronal depolarization also decreased the synthesis of lipids present in the myelin fraction. The relatively high specific radioactivity of myelin lipids and the results from both repeated purification experiments and mixing experiments ruled out the possibility that the radioactive lipids present in myelin could derive from contamination with other subcellular fraction(s). Because neither mature oligodendrocytes nor myelin is known to express voltage-dependent Na⁺ channels, it is conceivable that the effect of veratridine on myelin glycerolipid metabolism occurs by an indirect mechanism such as an increase in the extracellular [K⁺]. However, the presence of 60 mM KCl in the medium did not affect the acylation of either brain or myelin lipids. These results raise questions as to the absence of sodium channels in myelinating oligodendrocytes and/or myelin.     

In vitro incorporation of ( 3 H) methyl choline into phosphatidyl choline of rat liver subcellular fractions

Incubation of a rat liver total homogenate with radioactive choline and subsequent isolation of subcellular fractions, at different times, showed similar patterns of labeling. Incubation of microsomes, mitochondria and purified nuclei isolated from rat liver, showed that all fractions were able to incorporate the precursor into phosphatidyl choline. The specific activity was higher in mitochondria and increased in all cases with added supernatant. The addition of microsomes to mitochondria diminished the incorporation of label. Contamination of mitochondria by microsomes, was negligible as shown by undetectable amounts of cytochrome P₄₅₀, while NADPH₂ cytochrome c reductase showed a 10% contamination. A certain amount of radioactivity was incorporated in the absence of ATP and oxidizable substrates due to the presence of substrates and cofactors in the fraction and/or the supernatant. Labeled fractions reincubated with unlabeled choline, showed no loss of radioactivity, proving that incorporation was not due to simple exchange processes. It is concluded that although rat liver mitochondria can acquire part of their own provision of phosphatidyl choline by transference from microsomes, all organelles and specially mitochondria, can independently synthesize this phospholipid.     

Incorporation and distribution of dihomo-γ-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

Human keratinocytes in culture were labelled with ¹⁴C-dihomo-γ-linolenic acid, ¹⁴C-arachidonic acid or ¹⁴C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neural lipids a substantial amount, and as free unesterifed fatty acids only a minor amount. The most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-γ-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerosl as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.     

Lipid metabolism in myelinating glial cells: lessons from human inherited disorders and mouse models

The integrity of central and peripheral nervous system myelin is affected in numerous lipid metabolism disorders. This vulnerability was so far mostly attributed to the extraordinarily high level of lipid synthesis that is required for the formation of myelin, and to the relative autonomy in lipid synthesis of myelinating glial cells because of blood barriers shielding the nervous system from circulating lipids. Recent insights from analysis of inherited lipid disorders, especially those with prevailing lipid depletion and from mouse models with glia-specific disruption of lipid metabolism, shed new light on this issue. The particular lipid composition of myelin, the transport of lipid-associated myelin proteins, and the necessity for timely assembly of the myelin sheath all contribute to the observed vulnerability of myelin to perturbed lipid metabolism. Furthermore, the uptake of external lipids may also play a role in the formation of myelin membranes. In addition to an improved understanding of basic myelin biology, these data provide a foundation for future therapeutic interventions aiming at preserving glial cell integrity in metabolic disorders.     

Growth of animal cells on glass fiber filters and their utilization for biosynthetic analysis.

HeLa and L-M cells can be effectively grown directly on glass fiber filters to yield replicate cultures that allow easy analysis of biosynthetic capabilities through measurement of radioactive precursor uptake and incorporation. The glass fiber filters are superior to glass cover slips, growth in scintillation vials, and growth on Millipore filters in the ease of handling during experimental treatment and in the amount of radioactivity incorporated during the labeling period. These parameters are experimentally established and a typical application of the procedure that demonstrates the hydroxyurea inhibition of DNA synthesis is presented.     

Direct evidence of the role of propionate in choline synthesis in rats

Wistar rats were injected with 2-14C-propionate in a dose of 30 mu Ci/100 g bw, 2 h after food intake. Two hours after isotope injection the rats were decapitated to determine specific radioactivity (SR) in liver and brain lipids, in liver phosphatidylcholine (PC) and its structural components. The label was incorporated in liver lipids in a far greater amount. In liver PC, SR appeared the highest in glycerin and less higher in the fraction of higher fatty acids. The least amount of the label from 2-14C-propionate was incorporated in choline. The fact of the label incorporation in choline was recorded for the first time.     

Turnover of phosphatidylcholine in cell membranes of adult rat brain

—Incorporation of radioactive choline into subcellular fractions of adult rat brains, after intracranial injection of the precursor, was studied at different times. Maximum labelling of phosphatidylcholine occurred at 5 h in all fractions, the highest specific radio-activity being found in mitochondria. The specific radioactivity diminished rapidly for a few hours after the peak of maximum incorporation and continued to decrease thereafter at a much slower rate. The type of decay curve found in all fractions except myelin, indicated the possible presence of two populations of phosphatidylcholine with quite different turnover rates. The significance of these findings is discussed.