The identity of a myelin-like fraction isolated from developing brain

1. A myelin-like membrane fraction was isolated from developing rat brain by a new method. 2. The chemical composition and morphology of the fraction are described. 3. The myelin-like fraction is similar to myelin in characteristic enzyme activity but differs in the absence of basic protein and cerebrosides. No similarity to other subcellular fractions was observed. 4. It is suggested that the myelin-like fraction is a stage in the formation of compact myelin from glial plasma membrane. 5. ;Early' myelin consists of the myelin-like and compact myelin fractions from developing brain.     

Investigations on myelinogenesisin vitro: I. The occurrence of endogenous protein kinase and its role in the phosphorylation of myelin basic proteins in “Myelin-like membranes” isolated from cerebral cell cultures

The presence of a protein kinase capable of phosphorylating endogenous as well as exogenously added myelin basic proteins has been demonstrated in a myelin-like membrane fraction isolated from reaggregating and surface adhering, primary cultures of cells dissociated from embryonic mouse brain. Only the large and small components of myelin basic proteins were found to be phosphorylated when myelin-like membrane fraction was incubated with [-³²P]ATP. The protein kinase endogenous to the myelin-like membrane fraction was mainly of the cyclic AMP independent type. There was very little cyclic AMP dependent or cyclic GMP dependent protein kinase activities in this myelin-like fraction. Although the myelin basic proteins were the only endogenous proteins phosphorylated, protein kinase of the myelin-like membrane was capable of catalyzing the phosphorylation of exogenous substrates, such as histones.     

Calcium ion stimulated endogenous protein kinase catalyzed phosphorylation of basic proteins in myelin subfractions and myelin-like membrane fraction from rat brain

Polypeptide composition and endogenous phosphorylation were investigated in the subfractions of rat brain myelin isolated by either discontinuous or continuous sucrose density gradient centrifugation of myelin. Similarly, a myelin-like membrane fraction (SN4) was also studied. Observations were made that strongly indicated the presence of a calcium-stimulated protein kinase in a highly purified myelin preparation and which exclusively phosphorylated myelin basic proteins of the membrane preparation. Adenosine cyclic 3',5'-phosphate (cAMP) stimulated kinase on the other hand was found to be considerably enriched in the myelin-like membrane fraction. Although this latter enzyme is also capable of phosphorylating the basic proteins, its effect was at least 5 times weaker compared to the calcium-stimulated myelin protein kinase. Within the gradient subfractions there appeared a close relation between the amount of basic proteins and their calcium-stimulated phosphorylation; a similar relationship, however, was not obtained in the case of cAMP-dependent phosphorylation of myelin basic proteins. The former (i.e., Ca2+-stimulated phosphorylation) was found to require a protein factor that functionally resembled calmodulin. The results thus raises an interesting possibility of the presence of calmodulin-like proteins and a calcium-stimulated protein kinase in adult myelin membrane from mammalian brain, both of which have been hitherto unrecognized constituents of myelin membranes.     

Ontogenetic study of a myelin-derived fraction with 2'':3''-cyclic nucleotide 3''-phosphodydrolase activity higher than that of myelin.

A membrane fraction (SN 4), prepared from developing and mature rat forebrain by hypo-osmotic treatment of microsome (microsomal-fraction)-free myelin, appears to be closely related to the myelin-like fraction. Fraction SN 4 shows 2':3'-cyclic nucleotide 3'-phosphohydrolase activity higher than that of the parent myelin. Electrophoresis reveals a multitude of bands with the Wolfgram protein as the main component.     

Isolation and Characterization of Multilayered Sheath Membrane Rich in Glucocerebroside from Shrimp Ventral Nerve

A membrane fraction rich in glucocerebroside was isolated from homogenates of ventral nerves of pink shrimp (Penaeus duorarum) by sucrose gradient centrifugation. The membrane fraction was observed at 0.15 M sucrose and was rich in lipids (lipid/protein ratio approximately 15:1). Electron microscopy showed that the fraction was derived from myelin-like multilayered glial membrane ensheathing axons, which has morphological similarities to myelin. Most of the lipids in shrimp nerve, including glucocerebroside, sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and ethanolamine-plasmalogen, as well as cholesterol, appeared to be concentrated in this fraction. The fatty acids of these phospholipids were exclusively saturated or monounsaturated with C14-C26 chain lengths. The aldehyde moiety of plasmalogens contained only saturated C14-C18 carbon chains. Like glucocerebrosides, the sphingoid base of sphingomyelin consisted mainly of C14-C16 sphingenines and sphinganines, but they also contained significant amounts of C19 and C20 sphinganines. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins in this fraction showed several bands in the 23,000-85,000 Mr range. Radioimmunoassay, however, did not show cross-reactivity with antibodies to myelin basic protein. The functional role of this membrane in relation to mammalian myelin is discussed.     

2',3'-cyclic nucleotide-3'-phosphodiesterase in the central nervous system is fatty-acylated by thioester linkage

2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNP1 and CNP2 with Mr of 46,000 and 48,000, respectively) is the major enzyme of central nervous system myelin. It is associated with oligodendroglial plasma membrane and uncompacted myelin (myelin-like fraction), which are in contact with glial cytoplasm. Proteins of the myelin-like fraction were labeled with [3H]palmitic acid in brain slices from 17-day-old rats and immunoprecipitated with anti-CNP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material revealed intense acylation of CNP1 and CNP2, and radioactivity was released by hydroxylamine. Palmitic acid was covalently bound to CNP because radioactivity was not removed by extraction of immunoprecipitated CNP with organic solvent or by boiling in sodium dodecyl sulfate and dithiothreitol. However, treatment of immunoprecipitated CNP with (a) hydroxylamine-released palmitohydroxamate and palmitic acid, (b) sodium borohydride-released hexadecanol, and (c) methanolic-KOH-released methyl palmitate. Synthesis, acylation, or transport of CNP was not affected by monensin or colchicine. However, acylation of CNP was inhibited 24-32% by cycloheximide. These results provide conclusive evidence that CNP1 and CNP2 are fatty acid acylated with palmitate through a thioester linkage and is posttranslationally modified sometime after synthesis.     

Effects of monensin on posttranslational processing of myelin proteins

Rat brain slices were incubated with [3H]palmitic acid and [14C]glycine to label the lipid and protein moieties, respectively, of myelin proteolipid protein (PLP). The effects of monensin on posttranslational processing of proteins were examined by measuring the appearance of [14C]glycine- and [3H]palmitate-labeled proteins in myelin and myelin-like fractions. At 0.01 and 0.10 microM, monensin did not appreciably affect total lipid or protein synthesis; higher concentrations caused increased inhibition. Monensin at 0.10 microM markedly decreased the appearance of [14C]glycine-labeled PLP in myelin, but had little effect on the 14C basic proteins or the incorporation of [3H]palmitic acid into total or myelin PLP. The same relative effect was apparent at higher monensin concentrations. In the myelin-like fraction, monensin at 0.10 microM also depressed entry of [14C]glycine into protein comigrating with PLP, and again had no effect on incorporation of [3H]palmitic acid. In addition, monensin increased the [3H]palmitate label associated with two high-molecular-weight proteins in the myelin-like fraction with no concomitant increase in [14C]glycine label.     

Neurochemical Characteristics of Myelin-like Structure in the Chick Retina

Abstract: Certain characteristics of myelin-like structures in the chick retina were examined morphologically and biochemically. Developmental changes of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) in the chick retina and optic nerve were examined. The measurable activity in the retina was first detected at 16 days of incubation and thereafter, it increased rapidly until 4 weeks post-hatching. By contrast, CNPase activity in the optic nerve reached the maximum level at 4 days post-hatching and maintained a constant level thereafter. The purifed myelin fraction from the chick retina showed higher activity of CNPase, whereas its activity in the retinal homogenate was very low. Hence, it was considered that the myelin fraction from the chick retina is similar to that of CNS myelin with respect to CNPase. Protein profiles of the purified myelin fractions isolated from the chick optic tectum, optic nerve, retina and sciatic nerve were analysed by SDS-polyacrylamide gel elec-trophoresis. Myelin fractions from the chick optic tectum and optic nerve contained basic protein (BP) and Folch-Lees proteolipid protein (PLP). Myelin fraction from the chick sciatic nerve contained BP, P2 and two glycoproteins (PO and 23K). In contrast, retinal myelin fraction contained only BP. PLP, PO, 23K and P2 proteins were definitely undetectable. Electron micrographs revealed that some axons in the optic nerve fiber layer of the chick retina were wrapped by a spiral-structured myelin-like sheath, which showed some differences from those of CNS and PNS myelin sheaths. It was suggested that the origin of the myelin-like structure in the chick retina is other than from oligodendroglia or Schwann cells.     

Further studies on the smooth-surfaced membranes isolated from rat brain

The light and heavy smooth-surfaced membranes (LSM and HSM), which had densities corresponding to 1.08 M and 1.28 M sucrose, respectively, were isolated from rat brain and some of their biochemical properties were investigated. Both LSM and HSM showed high Na+,K+-ATPase activity and, in particular, in HSM the activity was four times (21.55 mumol/mg protein/h) higher than that of the brain homogenate. High 2',3'-cyclic nucleotide 3'-phosphodiesterase activity (293.4 mumol/mg protein/h) was characteristic of LSM. 5'-Nucleotidase and acetylcholinesterase activities were also higher in LSM than in HSM. SDS-polyacrylamide gel electrophoresis showed that LSM and HSM had many protein component and that low molecular weight proteins such as proteolipid protein and basic protein were almost absent, in contrast with myelin and myelin-like membrane. GM1 ganglioside constituted the major class of total ganglioside in both LSM and HSM. These biochemical findings suggested that LSM is a membrane that has not previously been described, or a membrane fraction related to the oligodendroglial plasma membrane.     

The isolation and characterization of a plasma membrane and a myelin fraction derived from oligodendroglia of calf brain.

—A method is described for the fractionation of bulk isolated oligodendroglial cells from calf brain to produce both a plasma membrane and an attached myelin fraction. The cells are homogenized in a sucrose solution containing Mg²⁺ and K+ at a pH of 6·5. Crude membrane fractions are obtained from this homogenate by discontinuous sucrose density gradient centrifugation. After being subjected to osmotic shock, these fractions are purified by continuous sucrose density gradient centrifugation. The plasma membrane fraction, which bands at 1·0 m -sucrose, was identified by its morphology and enzyme content. Electron microscopy showed it to be a homogeneous preparation of vesicles composed, for the most part, of smooth trilaminar membranes. Enzymatic analysis revealed the presence of high specific activities of Na+, K+-ATPase, 5′-nucleotidase and 2′,3′-cyclic AMPase. Lipid analysis showed a higher galactolipid and lower phospholipid content than has been reported for neuronal and synaptic membranes. The attached myelin fraction, which bands at 0·7 m -sucrose has the typical multilamellar appearance of myelin, but differs considerably from normal myelin in having high concentrations of plasma membrane marker enzymes, and a lipid composition intermediate between normal myelin and the plasma membrane fraction. The ganglioside content and protein patterns of these fractions have also been examined.     

CHANGES IN THE PROTEIN COMPOSITION OF MOUSE BRAIN MYELIN DURING DEVELOPMENT

Abstract— Myelin was isolated from the brains of mice at various ages by a procedure involving a final purification on a continuous CsCl gradient. Myelin protein accumulated throughout development, increasing from 0.25 mg of protein/brain at 8 days of postnatal age to 3.5 mg of protein/brain at 300 days, although the rate of accumulation was greatest at about 21 days of age. Quantitative studies of the protein composition of these samples were carried out, utilizing discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Mouse brain myelin, contained (in order of increasing molecular weight) two basic proteins, an uncharacterized doublet, proteolipid protein, and a group of high molecular weight proteins. There were marked changes in the quantitative distribution of these proteins with increasing postnatal age. The basic protein fraction of total myelin protein increased from about 18 per cent at 8 days to 30 per cent at 300 days of age. Proteolipid protein increased even more dramatically, from 7 to 27 per cent in the same time interval. These chemical studies were correlated with ultrastructural investigations, both of the developing myelin sheath in situ and the isolated myelin obtained from mice of various ages. A hypothesis, relating the observed changes in protein composition of myelin during development to its mode of formation, is developed. Another subcellular fraction, separated from myelin, by virtue of its greater density in a CsCl gradient, was also studied. It was a vesicular, membranous fraction present at a level of 0.35 mg of protein/brain at all ages and was related to myelin in terms of protein composition.     

Shaping membrane interfaces in lipid vesicles mimicking the cytoplasmic leaflet of myelin through variation of cholesterol and myelin basic protein contents

Myelin basic protein (MBP) is an intrinsically disordered protein and in the central nervous system (CNS) mainly responsible for connecting the cytoplasmic surfaces of the multilamellar, compact myelin. Increased posttranslational modification of MBP is linked to both, the natural development (from adolescent to adult brains) of myelin, and features of multiple sclerosis. Here, we study how a combination of this intrinsically disordered myelin protein with varying the natural cholesterol content may alter the characteristics of myelin-like membranes and interactions between these membranes. Large unilamellar vesicles (LUVs) with a composition mimicking the cytoplasmic leaflet of myelin were chosen as the model system, in which different parameters contributing to the interactions between the lipid membrane and MBP were investigated. While we use cryo-transmission electron microscopy (TEM) for imaging, dynamic light scattering (DLS) and electrophoretic measurements through continuously-monitored phase-analysis light scattering (cmPALS) were used for a more global overview of particle size and charge, and electron paramagnetic resonance (EPR) spectroscopy was utilized for local behavior of lipids in the vesicles' membranes in aqueous solution. The cholesterol content was varied from 060 % in these LUVs and measurements were performed in the presence and absence of MBP. We find that the composition of the lipid layers is relevant to the interaction with MBP. Not only the size, the shape and the aggregation behavior of the vesicles depend on the cholesterol content, but also within each membrane, cholesterol's freedom of movement, its environmental polarity and its distribution were found to depend on the content using the EPR-active spin-labeled cholesterol (CSOSL). In addition, DLS and EPR measurements probing the transition temperatures of the lipid phases allow a correlation of specific behavior with the human body temperature of 37 °C. Overall, our results aid in understanding the importance of the native cholesterol content in the healthy myelin membrane, which serves as the basis for stable and optimum protein-bilayer interactions. Although studied in this specific myelin-like system, from a more general and materials science-oriented point of view, we could establish how membrane and vesicle properties depend on cholesterol and/or MBP content, which might be useful generally when specific membrane and vesicle characteristics are sought for.     

Retention of a dialkylphosphatidylcholine in myelin and other membranes

We describe an attempt to incorporate a metabolically inert phospholipid analog into animal membranes, especially myelin, in vivo, with the view of eventual long-term membrane modification or membrane engineering. A sonicated suspension of a mixture of [¹⁴C] phosphatidylcholine and its dialkyl analog, [³H] tetradecyloctadecano(1)phosphocholine, was injected into the brain of weanling rats. Samples were counted of whole brain, myelin, liver, and carcass, at intervals from 1 to 63 days, and the composition of the extracted labeled lipid was determined by thin-layer chromatography. Both lipid labels were found to be cleared from the body at similar rates, but while phosphatidylcholine was metabolized within a day, with the label appearing mainly in the phosphatidylethanolamine fraction and in nonpolar lipids, the dialkylphosphatidylcholine remained intact, with retention in myelin of a small but almost constant amount for a month. Ways will have to be found to enhance uptake of the lipids by the brain.     

Differential effect of colchicine upon the entry of proteins into myelin and myelin related membranes

Brain slices from 20 day old rats were incubated with radioactive aminoacids in the presence and absence of 500 M colchicine and the appearance of labeled proteins in myelin and in a myelin-like fraction (SN₄ fraction) was measured. In the presence of the inhibitor, the entry of proteolipid proteins was decreased to 55% in myelin and to 45% in SN₄ fraction with reference to control values while the entry of basic proteins and other minor protein components was unaffected in both fractions. The synthesis of proteolipid proteins was not affected by the presence of colchicine; moreover, a slight accumulation of these proteins was observed in microsomes. The results suggest that the microtubular system is involved in the transport of proteolipid proteins from their site of synthesis to their site of deposition and that the various types of myelin proteins follow different transport routes to enter into this special type of membrane.     

Biosynthesis and compartmentalization of Po,apolipoprotein A-I,and lipids in the myelinating chick sciatic nerve

Myelin deposition in developing chick sciatic nerve is associated with rapid synthesis of lipids, the major myelin protein Po and apo A-I, a major constituent of plasma lipoproteins. In order to understand possible roles of apo A-I in myelin assembly the synthesis and appearance of Po, apo A-I and lipids was studied in an intracellular fraction, an intralamellar fraction thought to be related to, or derived from, myelin and compact myelin from rapidly myelinating sciatic nerve of 1 day chicks. Incorporation with methionine or pulse-chase experiments indicated that initial synthesis of Po occurs in the intracellular fraction followed by movement to the intralamellar fraction and myelin. Incorporation of labelled oleate into phospholipids suggested that initial synthesis occurs in the intracellular and intralamellar fractions with slow movement to myelin. Incorporation of labelled galactose into cerebrosides suggested that initial synthesis occurs partially in myelin with slow loss from this fraction to the intralamellar fraction. However, incorporation of methionine into apo A-I indicated that initial synthesis occurred in the intracellular fraction with some transfer to the intralamellar fraction and secretion of a major portion into the incubation medium. It is concluded that the subcellular distribution of nascent apo A-I is not well coordinated with the distribution of other nascent constitutents of the myelin membrane. The accumulation of nascent Po, phospholipids and cerebrosides in the intralamellar fraction compared to compact myelin suggests that this fraction may play a role as a precursor membrane or as a storage site for assembly of myelin constituents into compact myelin.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - apo apolipoprotein - PC phosphatidylcholine - PE phosphatidylethanolamine     

Ratio and composition of the plasmalogen and diacylated forms of phospholipids in subcellular fractions of the avian brain

Studies have been made on the specific content of plasmalogen and diacylated forms of phosphatidylethanolamine and phosphatidylcholine in subcellular fractions (myelin, nuclei, microsomes, mitochondria, synaptosomes) from the brain of pigeons, as well as in the myelin fraction from the brain of the crow Corvus cornix and the hawk Accipiter gentelis. Fatty acid composition and fatty aldehyde composition of these two main phospholipids of the brain were studied in the subcellular fractions obtained. It was shown that plasmalogen forms of phospholipids are localized in birds mainly in the myelin fraction which exhibits the highest plasmalogen concentration as compared to the same fraction of all the vertebrates investigated. With respect to fatty acid and fatty aldehyde composition, as well as to the degree of their unsaturation, myelin plasmalogens from birds are similar to those from other cold-blooded and warm-blooded animals. This fact indicates that high relative content of plasmalogens together with their high unsaturation account for normal functional activity of myelin membranes in all vertebrates.     

RGD-containing peptides inhibit the synthesis of myelin-like membrane by cultured oligodendrocytes

A synthetic peptide derived from the fibronectin cell-binding domain, GRGDSP, inhibits the adhesion of rat oligodendrocytes to a number of substrates. However, while GRGDSP inhibited the adhesion of cells in a short term adhesion assay, the presence of the peptide did not prevent cells from adhering and thriving in longer term culture. The morphological characteristics of individual cells cultured with 0.1 mg/ml GRGDSP were similar to untreated cultures; small rounded cell bodies radiating numerous fine processes. Peptide-treated cultures were inhibited in their ability to produce myelin specific components. The characteristic developmental peak in sulfolipid synthesis which occurs both in vivo and in vitro was completely inhibited when cells were cultured with GRGDSP. In addition, the synthesis of myelin basic protein was inhibited. Ultrastructurally, cells treated with GRGDSP showed a greatly reduced number of multilamellar myelin-like membrane figures than cells grown without peptide or those grown with GRADSP. Cultured oligodendrocytes did not become sensitive to inhibition of sulfolipid synthesis by GRGDSP until a period immediately preceding the peak in sulfolipid biosynthesis. The effects of pretreatment with peptide for 5 d before this time were completely reversible. Pretreatment which extended into the time of peak myelin synthesis resulted in permanent impairment in the cell's ability to synthesize sulfolipid. The oligodendrocyte's ability to synthesize a myelin-like membrane in culture is, in part, inherent since it occurs in the absence of neurons. The present results indicate that myelin membrane production is also subject to external control since it appears that occupancy of an RGD-dependent cell surface receptor during a critical period of in vitro development is required for the oligodendrocyte to produce myelin-like membrane.     

Knockdown of Unconventional Myosin ID Expression Induced Morphological Change in Oligodendrocytes

Myelin is a special multilamellar structure involved in various functions in the nervous system. In the central nervous system, the oligodendrocyte (OL) produces myelin and has a unique morphology. OLs have a dynamic membrane sorting system associated with cytoskeletal organization, which aids in the production of myelin. Recently, it was reported that the assembly and disassembly of actin filaments is crucial for myelination. However, the partner myosin molecule which associates with actin filaments during the myelination process has not yet been identified. One candidate myosin is unconventional myosin ID (Myo1d) which is distributed throughout central nervous system myelin; however, its function is still unclear. We report here that Myo1d is expressed during later stages of OL differentiation, together with myelin proteolipid protein (PLP). In addition, Myo1d is distributed at the leading edge of the myelin-like membrane in cultured OL, colocalizing mainly with actin filaments, 2′,3′-cyclic nucleotide phosphodiesterase and partially with PLP. Myo1d-knockdown with specific siRNA induces significant morphological changes such as the retraction of processes and degeneration of myelin-like membrane, and finally apoptosis. Furthermore, loss of Myo1d by siRNA results in the impairment of intracellular PLP transport. Together, these results suggest that Myo1d may contribute to membrane dynamics either in wrapping or transporting of myelin membrane proteins during formation and maintenance of myelin.     

SIMULTANEOUS ISOLATION OF PURIFIED MICROSOMAL and MYELIN FRACTIONS FROM RAT SPINAL CORD

Abstract— The fraction that sediments between 2 × 10⁵ g -min and 6 × 10⁶ g -min from dilute dispersions of rat brain in 0.32 m -sucrose is a microsomal fraction with very little contamination by myelin. A crude microsomal fraction prepared in the same way from rat spinal cord contains more myelin than microsomes. Centrifugation of the crude microsomal fraction in 0.85 m -sucrose gave a floating fraction, an infranatant fraction (purified microsomes) and a small pellet. The purified microsomes contained very little myelin as judged by electron microscopy and polyacrylamide gel electrophoresis. The lipid composition resembled that of spinal cord myelin except that the purified microsomes contained relatively less cholesterol and ethanolamine plasmalogens. The content of galactolipids was much greater in spinal cord microsomes than in brain microsomes. The spinal cord CDP-ethanol-amine:diglyceride ethanolaminephosphotransferase activity (EC 2.7.8.1) was concentrated in the purified microsomes.
A spinal cord myelin fraction isolated from the 2 × 10⁵ g -min pellet was quite pure as judged by electron microscopy, enzyme activities and polyacrylamide gel electrophoresis. No NADPH-cyto-chrome c reductase activity (EC 1.6.2.3) could be detected in the purified myelin. The ethanolaminephosphotransferase specific activity was about 5% of that found in the purified microsomal fraction. The protein content was 25% by weight for spinal cord myelin and 31% for brain myelin. Of the total spinal cord 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, 16% was lost from the crude myelin during purification, 21% was recovered in the purified myelin, and 11% was found in the floating fraction from the crude microsomes. The purified myelin and microsomal fractions from spinal cord were relatively pure. Additional myelin was recovered in the floating fraction from the crude microsomes.     

Lipid composition of human cerebral white matter and myelin in phenylketonuria

Abstract— White matter and purified myelin from cerebral tissue obtained at autopsy from four phenylketonuric and five non-phenylketonuric mentally-retarded patients were analysed for lipids, DNA and protein. The lipid composition of the white matter and myelin was compared with that of a representative non-myelin component of white matter, the crude mitochondrial fraction. The total lipid content was significantly lower and the ratio of cholesterol to galactolipid was significantly higher in the white matter from the PKU patients than in that from the non-PKU patients. The lipid compositions of the myelin and ‘mitochondrial’ fraction, although differing from each other, did not exhibit appreciable differences between the PKU and non-PKU brain samples. However, the amount of myelin recovered from the brains of the PKU patients was, on the average, 40 percent lower than that recovered from non-PKU brains. The abnormal cholesterol: galactolipid ratio of PKU white matter could be accounted for by the altered proportion of myelin to non-myelin lipid components. The finding in PKU brains of a normal composition of lipids in the purified myelin and the absence of cholesterol esters in the white matter suggest that the deficiency in myelin may reflect an early arrest of myelination.