Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis.

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca2+. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.     

ALTERATION OF MYELIN BIOSYNTHESIS IN SLICES OF RABBIT SPINAL CORD BY ANTISERUM TO MYELIN BASIC PROTEIN AND BY PUROMYCIN 1

Abstract— Slices of rabbit spinal cord were incubated with [³H]tyrosine and [³⁵SO₄] in the presence of either 5% antiserum to myelin basic protein or 0.21 mM-puromycin. The degree of incorporation of the precursors into the basic protein (BP), the proteolipid protein (PLP) and into sulphatides, as a representative lipid, in isolated myelin was investigated. Anti-BP serum inhibited the incorporation of [³H]tyrosine into BP and PLP from 22 to 46% as compared to controls, whereas puromycin nearly completely inhibited incorporation. The incorporation of [³⁵SO₄] into sulphatides was inhibited by anti-BP serum from 20 to 34% and by puromycin from 33 to 65% as compared to controls. These alterations were myelin-specific as shown by the equal or even increased incorporation of the precursors into the homogenates of spinal cord. The results are discussed in relation to the interaction of lipids and proteins in membrane assembly.     

Neurotransmitter movements in nerve endings. Influence of substances that modify the interfacial potential

Polysialogangliosides, sulphatides, glycerylmonooleate, unsaturated fatty acids, myelin basic protein and sucrose inhibit the Na+-coupled uptake and induce a Ca2+-dependent release of dopamine from nerve endings. Substances chemically related to those referred to above, such as monosialogangliosides, neutral glycosphingolipids, glycerylmonostearate, saturated fatty acids and albumin, do not show these effects. Mixtures of polysialogangliosides or sulphatides with myelin basic protein or albumin inhibit, to different degrees, the effects of the individual components. The decreased uptake induced by sucrose reverted to control levels upon reduction of the concentration of the perturbing agent. The restoration of the uptake was probably mediated by the Na+-pump reconstituting the transmembrane Na+-gradient necessary for the Na+-coupled cotransport of dopamine. It is suggested that the effects of uptake inhibitor or release inducer agents derive from their ability to decrease the surface potential and modify the molecular organization of phospholipid interfaces which can result in changes of the membrane ionic permeability.     

X-ray diffraction and electron microscope study of the interactions of myelin components. The structure of a lamellar phase with a 150 to 180 A repeat distance containing basic proteins and acidic lipids

A variety of phases has been studied: those formed by lipids extracted from myelin, the basic myelin proteins A1 (from the central nervous system) and P1 (from the peripheral nervous system) or other basic proteins. A particularly interesting type of phase was observed which consists of one of the basic proteins of myelin, acidic phospholipids and sulphatides; this phase is lamellar and contains two lipid bilayers in its unit cell. The structure of this phase was determined by the pattern recognition technique and by electron microscope observations of OsO₄-flxed and freeze-etched preparations. It is formed by two different lipid bilayers, one containing mainly the phospholipids with the hydrocarbon chains in a liquid-like conformation and the other containing mainly the sulphatides with at least one fraction of the chains stiff and hexagonally packed. Under the effect of high temperature, or if cholesterol is added, this phase is replaced by other phases which lack the large repeat. The segregation of the lipids and their specific associations with the basic proteins are discussed in relation to the structure of myelin.     

Interaction of soluble and membrane proteins with monolayers of glycosphingolipids.

1. The interactions of four proteins (albumin, myelin basic protein, melittin and glycophorin) with eight neutral or acidic glycosphingolipids, including sulphatides and gangliosides, five zwitterionic or anionic phospholipids and some of their mixtures, were studied in lipid monolayers at the air/145 mM-NaCl interface. 2. In lipid-free interfaces, the surface pressure and surface potential reached by either soluble or integral membrane proteins did not reveal marked differences. 3. All the proteins studied showed interactions with each of the lipids but the maximal interactions were found for basic proteins with acidic glycosphingolipids. 4. Surface-potential measurements indicated that different dipolar organizations at the interface can be adopted by lipid-protein interactions showing the same value for surface free energy. 5. The individual surface properties of either the lipid of protein component are modified as a consequence of the lipid-protein interaction. 6. In mixed-lipid monolayers, the composition of the interface may affect the lipid-protein interactions in a non-proportional manner with respect to the relative amount of the individual lipid components.     

A permeability change of myelin membrane vesicles towards cations is induced by MgATP but not by phosphorylation of myelin basic protein

The existence of an endogenous protein kinase activity and protein phosphatase activity in myelin membrane from mammalian brain has now been well established. We found that under all conditions tested the myelin basic protein is almost the only substrate of the endogenous protein kinase in myelin of bovine brain. The protein kinase activity is stimulated by Ca²⁺ in the micromolar range. Optimal activity is reached at a free Ca²⁺ concentration of about 2 μM. Myelin membrane vesicles were prepared and then shown to be sealed by a light-scattering technique. After preloading with ⁴⁵Ca²⁺, ⁸⁶Rb⁺, or ²²Na⁺, the self-diffusion (passive outflux) of these ions from myelin membrane vesicles was measured. Ionophores induced a rapid, concentration-dependent outflux of 80–90% of the cations, indicating that only a small fraction of the trapped ions was membrane bound. There was no difference in the diffusion rates of the three cations whether phosphorylated (about 1 mol phosphate per myelin basic protein) or non-phosphorylated vesicles were tested. In contrast, a small but significant decrease in permeability for Rb⁺ and Na⁺ was measured, when the vesicles were pretreated with ATP and Mg²⁺.     

Butanol extracts from myelin fragments. VI. Lack of specificity of 5-hydroxytryptamine binding to recombinate fraction with sulphatides and phosphatidylinositol

To clarify the participation of sulphatides and PI for the binding of 5-HT to myelin butanol extracts, binding experiments of ¹⁴C·5-HT to the recombinate fraction of these two acidic lipids were performed. The recombinate fraction was incubated with 5 × 10^?7 M of ¹⁴C·5-HT, and elution profile of ¹⁴C·5-HT, sulphatides and PI was examined by Sephadex LH₂₀ column chromatography. Three components were eluted with CM 4:1 and elution areas of those relatively corresponded to each other. On the other hand, when 3 fold excess volumes of solvents were used ¹⁴C·5-HT was also eluted with CM 4:1 but the clear difference was observed in the elution pattern of sulphatides and PI. In the myelin butanol extracts, we have reported that the 5-HT binding macromolecules present in the myelin extracts would be composed of saturable and non-saturable components, and ACh, DA and tryptamine specifically inhibited the saturable 5-HT binding. Therefore, on the recombinate system of two acidic lipids the specificity of ¹⁴C·5-HT binding was investigated by displacement experiments. The results indicated that only ACh slightly inhibited the ¹⁴C·5-HT binding but 5-HT, DA and tryptamine had no effect. All these observations suggest that other component(s) besides sulphatides and PI may be implicated in the binding of 5-HT to myelin butanol extracts.     

Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent

The localization of proteins in myelin was studied by the use of a non-penetrating penetrating reagent. Tritiated 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with ³H-labelled, 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

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.     

Preparation of membrane vesicles from isolated myelin. Studies on functional and structural properties

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in ²²Na⁺ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myelin basic protein from 0–150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

Protein kinase associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates

When highly purified myelin from rat sciatic nerve was incubated with [γ-³²P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and ³²P from [γ-³²P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg²⁺-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3′,5′-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing.From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

THE CHEMICAL COMPOSITION OF MYELIN IN ORGAN CULTURES OF RAT CEREBELLUM

Myelinating organ cultures of rat cerebellum were maintained in vitro for up to 130 days. Extensive myelination took place between 7 DIV (days in vitro) and 28 DIV. Centrifugation of a crude culture myelin fraction on a discontinuous gradient yielded three layers termed light myelin, heavy myelin and membrane fraction, which exhibited an ultrastructure virtually identical to that of comparable layers prepared from surviving littermates. However, culture myelin layers showed a gross deficiency of galactolipids with a relative increase in phospholipids. The 2,3′-cyclic nucleoside-monophosphate phosphodiesterase (CNP) activity was decreased in the culture myelin layers, but not to an extent comparable to the cerebroside deficiency. A form of “slow myelin maturation” takes place in vitro with both myelin cerebrosides and sulphatides increasing in cultures older than 60 DIV. The results indicate that CNS myelination comprises at least two phases, and that the second phase involving galactolipid enrichment of myelin can, under experimental conditions, be partly uncoupled from the first phase without affecting the morphology or ultrastructure of the sheaths.     

Myelin metabolism in vitro in experimental allergic encephalomyelitis

Abstract— Spinal cord slices from rats in different stages of allergic encephalomyelitis (EAE) were incubated with [U-¹⁴C]glucose. Normal rats and rats injected with Freund's adjuvant served as controls. The slices were fractionated by a discontinuous sucrose gradient into purified myelin and a heavy membrane residue, the lipids and proteins were extracted, and their specific activities were determined. Uptake of ¹⁴C into myelin lipids was depressed in the rats with acute EAE, while an increase was shown in myelin protein and heavy membrane lipids and proteins. The increased synthesis in non-myelin fractions was ascribed to invasion of metabolically active cells. The depression in myelin lipid synthesis occurred early in the disease before lesions appeared or the inflammatory reaction became widespread. Myelin from guinea pigs with acute EAE resulting from injection of a purified basic protein also showed a depression of uptake in both lipids and proteins. It is suggested that a metabolic insult as a result of the immunological process is dealt the oligodendroglial cells early in the course of the disease which leads to a weakening of the myelin sheath and subsequent phagocytosis of myelin.     

Endogenous cyclic AMP-stimulated phosphorylation of a Wolfgram protein component in rabbit central-nervous-system myelin.

Cyclic AMP-stimulated phosphorylation of membrane proteins in central-nervous-system myelin was investigated, with rabbit brain myelin. Subfractionation of a myelin membrane preparation by sucrose-density-gradient centrifugation produced a rapidly sedimenting population of membrane vesicles containing 5'-nucleotidase and acetylcholinesterase, a light membrane fraction containing myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase, and an intermediate membrane fraction containing the highest specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase and a small proportion of myelin basic protein. Cyclic AMP stimulation of protein phosphorylation was confined to a protein of Mr 49 700, which co-electrophoresed with the upper component of the Wolfgram protein doublet. Cyclic AMP did not affect the phosphorylation of myelin basic protein. Cyclic AMP-stimulated phosphorylation of this protein followed 2',3'-cyclic nucleotide 3'-phosphodiesterase activity on subcellular fractionation and was correspondingly high in the intermediate or 'myelin-like' fraction on sucrose-density-gradient centrifugation.     

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.     

Neutral protease activity in lymphocytes of lewis rats with acute experimental allergic encephalomyelitis

Lymphocytes from popliteal and inguinal lymph nodes of Lewis rats with acute EAE as a result of injection of lyophilized guinea pig myelin in Freund's complete adjuvant exerted strong proteolytic activity at neutral pH toward myelin basic protein. After injection of myelin the level of proteolytic activity remained about the same as that in lymphocytes from Freund's adjuvant-injected controls until about day 10 after injection, just before the onset of paralytic symptoms; then the proteolytic activity increased to approximately double its former level. Myelin basic protein was hydrolyzed by whole lymphocytes, but more activity was unmasked by homogenization. Similar results were also obtained using lymphocytes from thymus of EAE and control animals. Lymphocytes with high levels of proteolytic activity were not absorbed by glass wool, did not stain with neutral red, nor did they phagocytose antibody-coated sheep red blood cells. Thymus and lymph node lymphocytes cleaved myelin basic protein to three major peptides and a fourth minor peptide, while spleen lymphocytes hydrolyzed basic protein at only one point resulting in two peptides whose molecular weights added up to that of myelin basic protein. The protease activity was inhibited by 5×10^–3 Mp-chloromercuribenzoate and by phenylmethyl sulfonyl fluoride, TPCK, and soybean trypsin inhibitor, therefore the enzymatic activity probably depends on a serine residue and a sulfhydryl group. The bulk of the enzymatic activity is mostly membrane bound with the highest specific activity and total activity contained in a lysosomal-mitochondrial fraction. In view of the infiltration of lymphocytes into the brain substance in acute EAE, it is suggested that these cells may contribute to the destruction of myelin which is usually attributed to the monocyte or macrophage.     

The polyadenylated RNA directing the synthesis of the rat myelin basic proteins is present in both free and membrane-bound forebrain polyribosomes.

Free and membrane-bound polyribosomes were isolated from the forebrain of actively myelinating 24-day-old rats. The poly(A)+ RNA (polyadenylated RNA) extracted from both fractions was translated in vitro in reticulocyte lysates [Hall & Lim (1981) Biochem. J. 196. 327-336] in the presence or absence of a heterologous microsomal membrane fraction from dog pancreas. The rat myelin basic proteins synthesized in vitro were isolated by CM-cellulose chromatography and by immunoprecipitation with purified anti-(myelin basic protein) antibody. The large (mol.wt. 18 500) and small (mol.wt. 16 000) myelin basic proteins were translational products of poly(A)+ RNA from both free and membrane-bound polyribosomes. The identity of the myelin basic proteins was verified by analysis of peptides generated by the cathepsin D digestion of the immunoprecipitated proteins synthesized in vitro, in comparison with authentic rat myelin basic proteins. Although several other translational products of membrane-bound polyribosomal poly(A)+ RNA were modified when microsomal membranes were present during translation, molecular weights of the myelin basic proteins themselves were unchanged. The myelin basic proteins synthesized in vitro also did not differ significantly in size from the authentic myelin basic proteins, indicating that these membrane proteins are unlikely to be synthesized as substantially larger precursor molecules. The presence of the specific mRNA species on both free and membrane-bound polyribosomes is compatible with the extrinsic location of the myelin basic proteins on the cytoplasmic surface of the myelin membrane.     

LACK OF PHOSPHOLIPID TRANSPORT MECHANISMS IN CELL MEMBRANES OF THE CNS1

The possible transport role of phospholipid-protein complexes, present in the cell supernatant of rat brain was investigated using labelled choline as precursor of phosphatidyl choline. Results obtained after the intracranial injection of choline gave no indication of a sequence of events compatible with a transport of phospholipid molecules from the possible site of synthesis (microsomes) to the supernatant and subsequently to myelin. Chase experiments using rat brain slices incubated in vitro with radioactive choline agreed well with the above mentioned results. Contrariwise, when Na³⁵₂SO₄ was used as precursor, the results clearly indicated that synthesis of sulphatides takes place in microsomes, followed by transfer of the radioactive lipid to sulphatide-containing lipoproteins in the supernatant and finally to myelin. Results presented in this paper seem to give further support to the idea that other subcellular fractions, besides microsomes, can autonomously synthesize part of their own provision of phospholipids. Possible reasons which might explain the marked differences between the mechanisms of addition of phospholipids and sulphatides to myelin are discussed in relation to results obtained by other investigators.     

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [³²P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [³H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [³H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.     

PROTEOLIPID PROTEIN: SYNTHESIS AND ASSEMBLY INTO QUAKING MOUSE MYELIN

The incorporation of radioactive glycine into the major myelin proteolipid protein isolated from whole brain and from purified myelin of Quaking mice and normal littermates was compared. In a typical experiment, four Quaking mice and four littermate controls were injected intracranially with 250 μCi [2-³H]glycine and 25 μCi [U-¹⁴C]glycine respectively. Three hours later, the eight mice were killed and their brains combined. Equivalent portions were taken for (1) chloroform-methanol (2:1) extraction followed by ether precipitation of proteolipid from the brain and (2) myelin preparation. The ^3H/¹⁴C ratios for the microsomes:, the major myelin proteolipid as well as the other non-myelin proteolipids extracted from whole brain was approx 3.0. while the ³H/¹⁴C ratio for proteolipid protein in myelin was near 0.4. These findings were consistent for ages studied between 18 and 90 days. The results indicate that the synthesis of the major myelin proteolipid protein in the whole brain of Quaking mouse, as seen previously in our studies on basic protein, proceeds at a normal rate relative to microsomes but its incorporation into myelin is depressed. A working hypothesis of myelin membrane assembly is presented to account for the defect in the incorporation of these proteins into Quaking myelin.