H. Dreyfus

DEVELOPMENTAL PATTERNS OF GANGLIOSIDES AND OF PHOSPHOLIPIDS IN CHICK RETINA AND BRAIN

Abstract— The changes in phospholipids and gangliosides during ontogenesis of chick retina have been compared with those in brain. Three phases of accumulation of ganglioside NeuNAc in the retina were detected. In contrast, brain NeuNAc rapidly increased during embryonic life until hatching, followed by a slower increase up to the adult stage. The phospholipid changes in retina and in brain occur in a‐similar manner to the variations observed for gangliosides, however in retina the changes of phospholipid content are less marked than in brain, during embryonic life. There were marked changes in the retina and brain ganglioside patterns with age. Gd3 and Gd1b decreased rapidly in per cent; correspondingly, Gd1a increased during embryonic life and became the major ganglioside in place of Gd3. There was a similarity between ganglioside patterns of chick retina and brain. Except for some slight variations during embryonic life, the retinal phospholipid pattern did not change noticeably.

Direct thin-layer chromatography of gangliosides of a total lipid extract.

A new method of thin-layer chromatography of gangliosides has been developed. The method does not need the preliminary purification of the gangliosides so that a total lipid extract can be applied to a precoated silica gel plate. Under these conditions, gangliosides are not lost as is the case in other procedures involving ganglioside partition in a water phase, dialysis, hydrolysis, etc. Monodimensional ascending chromatography is performed in a sandwich chamber placed in a tank protected from draughts and temperature variations (24 ± 1°C). Three successive solvent systems are used: (1) chloroform; (2) chloroform-methanol-water (70-30-4, v/v/v); (3) chloroform-methanol-0.25% KCl (60-35-8, v/v/v). The procedure has been used for the ganglioside distributions of chicken brain and retinal gangliosides as well as for different brain areas of an inbred mouse strain. The brain and retinal gangliosides pattern are similar to those obtained with purified gangliosides. GD1a in the major ganglioside and appreciable amounts of GM1, GD3, GD1b, and GT1 have been found. The whole ganglioside pattern of cerebellum, forebrain, hippocampus, pons and medulla and the remaining brain are given. The ganglioside distribution differs among the different brain regions with the predominance of either GD1a or GT1.

Enzymatic synthesis of lactosylceramide by a galactosyltransferase from developing chicken retina

Abstract The activity of UDP-galactose: glucosylceramide galactosyltransferase was studied during chick retina ontogenesis. This galactosyltransferase catalyses the formation of lactosylceramide, a reaction which is considered to be one of the initial steps of ganglioside biosynthesis. The activity was detected in 7 day-old embryos and was present in adult animals. The highest specific activity was found at the 11th day of embryonic life. The radioactive product was identified as lactosylceramide by comparison with standard glycolipids, by radiochromatography, by thin layer chromatography of the o -methyl-glycoside derivatives and by gas-liquid chromatography. The enzyme required the presence of Mn 2+ , had a pH optimum of about 6·7 and was stimulated by Triton X-100.

Gangliosides and phospholipids of the membranes from bovine adrenal medullary chromaffin granules.

The lipid and ganglioside compositions of membranes of chromaffin granules isolated from bovine adrenal medulla have been investigated. The detailed lipid analysis revealed the presence of high levels of lysophosphatidylcholine, in agreement with previous studies, but also of sphingomyelin and plasmalogens. From these membranes, gangliosides have been extracted and separated by thin-layer chromatography and analysed. 95% of the total recovered gangliosides were hematosides (GM3), which migrated as three major species. Sugar analyses have been performed, as well as the fatty acid compositions. The three hematoside gangliosides appeared to differ on the basis of their fatty acid composition. Compared with the brain, chromaffin granule membranes showed a simple ganglioside composition, thus offering a good model for the study of the metabolism and the role of gangliosides. The simple ganglioside composition of chromaffin granule membranes has allowed us to state that there are 60 mol phospholipid and 30 mol cholesterol per mol ganglioside.

A study on the topological distribution of phospholipids in microsomal membranes of chick brain using phospholipase C and trinitrobenzenesulfonic acid.

The transbilayer distribution of phospholipids in chicken brain microsomal membranes has been investigated using trinitrobenzenesulfonic acid and phospholipase C from Clostridium welchii. The exposure of intact microsomes to trinitrobenzenesulfonic acid showed that the labelling of aminophospholipids followed biphasic kinetics, indicating that these membranes contain a fast- and a slow-reacting pool of aminophospholipids. Use of microsomes radioiodinated on their surface led to the conclusion that the fast-reacting pool may be located on the outer leaflet of the microsomal vesicles. It contains about 35% of the phosphatidylethanolamine, 29% of the ethanolamine plasmalogens and 18% of the phosphatidylserine. The treatment of intact microsomes with the phospholipase C Cl. welchii produced the hydrolysis of 50% of the phospholipids without any loss of their permeability properties, indicating that they are not permeable to the hydrolase. Phospholipids extracted from the microsomes were hydrolyzed rapidly by the phospholipase C with the exception of phosphatidylserine and phosphatidylinositol. In intact microsomes about 90% of phosphatidylcholine, 32% of ethanolamine phospholipids and 60% of sphingomyelin were accessible to the phospholipase. These results suggest that the phospholipids have an asymmetric distribution in chicken brain microsomes, the external leaflet containing about 75% of the choline phospholipids and 25% of the aminophospholipids, whereas an opposite distribution is observed in the inner leaflet.

Synthesis of choline phospholipids in neuronal and glial cell cultures by the methylation pathway.

The synthesis of choline in the nervous tissue has been a subject of debate (reviews [1,2]). Since the work of [3,4] it was believed that the stepwise methylation of ethanolamine and/or phosphatidylethanolamine in nervous tissue was non-existant or irrelevant until the suggestion that choline might be produced de novo in the rat brain [5-7] through methylation of phosphatidylethanolamine. Methyltransferase activity has been shown in rat brain synaptosomes [8,9], suggesting that nervous tissue may have the necessary machinery for the de novo synthesis of phosphatidylcholine. The possibility of obtaining cell cultures containing exclusively neurons or glial cells gave us the opportunity to check, in these isolated systems, whether neurons and/or glia can methylate phosphatidylethanolamine to phosphatidylcholine. The results obtained suggest that both cell types have this capacity and that the synthesis of choline phospholipids through the methylation pathway is much higher in glial cells than in neurons.

Molecular species of choline and ethanolamine phospholipids in rat cerebellum during development

Abstract: The molecular species composition of rat cerebellar phospholipid subclasses has been studied by HPLC after phospholipase C treatment and dinitrophenyl derivatization. During rat cerebellum development (3–90 days postpartum), cholinephosphoglycerides and ethanolamine phosphoglycerides represented ∼80% of all phospholipids, with their relative amount changing after 1 month. Among ethanolamine phosphoglycerides, the molar ratio of diacylglycerophosphoethanolamine (diacylGPE) to alkenylacylGPE decreased from ∼1.4 at 3 days to ∼0.5 after 10 days. The phospholipids investigated contained up to 12 different molecular species. The rate of accumulation of the various molecular species of diacylglycerophosphocholine (diacylGPC), diacylGPE, and alkenylacylGPE during cerebellar development allowed a classification into three main groups. The overall increase of the molecular species of the first group (6‐diacylGPC, 5‐diacylGPE, and 4‐alkenylacylGPE) was ∼ 18‐fold between 3 and 90 days, with a faster rate of accumulation between 3 and 30 days. Those of the second group (3‐diacylGPC, 5‐diacyl‐GPE, and 5‐akenylacylGPE) increased by ∼45‐fold during the same developmental period, at a slow rate before day 15 and at a faster one thereafter. The molecular species of the third group (3‐alkenylacylGPE) increased by >250‐fold between 3 and 90 days, at a very slow rate before day 21 and more quickly thereafter. The different rates of accumulation of the components of the three groups during cerebellar development suggest a preferential location of the first group in membranes of neuronal perikaryons, glial cells, and synaptosomal structures. Those of the second group appear to be located in both synaptosomal membranes and myelin sheets, and those of the third group can be considered as myelin markers.

ENZYMATIC BIOSYNTHESIS OF ETHANOLAMINE- AND CHOLINE-PHOSPHOGLYCERIDES IN RETINA DURING DEVELOPMENT. EFFECT OF LIGHT STIMULATION

Choline‐ and ethanolamine‐phosphoglycerides (CPG and EPG) are the most abundant phospholipids of retinal membranes. We have investigated some regulatory mechanisms involved in the final steps of their biosynthesis, namely those catalysed by CDP‐choline 1,2 diradyl‐sn‐glycerol choline phosphotransferase (CPT) and CDP‐ethanolamine 1,2 diradyl‐sn‐glycerol ethanolamine phosphotransferase (EPT). We have studied both enzymes in the retina which offers an excellent model for the investigation of the molecular basis of the effect of its physiological stimulus, the light. In chick retina. the specific activity (SA) of EPT reached a maximum at the 18th day of embryonic life and decreased thereafter. In the case of CPT, a similar peak of SA was observed at hatching. The time of maximum SA of EPT and CPT corresponded to the period during which retinal rod outer segments are formed. The apparent Km values of EPT and CPT determined with whole retinal homogenates for CDP‐bases showed different profiles. The apparent Km of EPT decreased during embryonic life and increased thereafter whereas the apparent Km of CPT did not change during ontogenesis. Light stimulation of calf retinal homogenates had different effects on phosphotransferase activities. In the presence of only endogenous diacylglycerol (DAG) the SA of CPT was 2‐fold higher for dark‐adapted retinas, whereas no differences in EPT activities were observed. After addition of exogenous DAG (4mM) to the incubation medium, light stimulation of the retina led to a 50% increase of EPT activity whereas no effect was observed for CPT. These different effects could be related to the cyclic nucleotides present in retina before and after light stimulation. In addition all the data presented in this study indicate that, as in brain, CPT and EPT in retina are two different enzymes.

Possible role of sialocompounds in the uptake of choline into synaptosomes and nerve cell cultures

Incubation of primary nerve cell cultures and of crude synaptosomal preparations with neuraminidase released sialic acid from both gangliosides and sialoglycoproteins. After this treatment, the pattern of ganglioside distribution was severely modified with a decrease of polysialogangliosides (GD1b, GT1b, GT1L, GQ1) and a dramatic increase in monosialoganglioside GM1. The choline influx into neuraminidase treated cells and organelles was reduced by 30–50% but the efflux was unmodified. In particular the high affinity mechanism of choline uptake disappeared and the low affinity mechanism was modified in both cases. The disappearance of the high affinity uptake mechanism was not followed by a decreased acetylcholine synthesis as it should be if the current theories on choline uptake and acetylcholine synthesis are correct. Our present data thus confirm our previous hypothesis that choline metabolism regulates choline uptake rather than the other way round as is suggested by the theories most widely accepted at present. Choline uptake was unaffected by pretreatment of cells and organelles with tetanus toxin suggesting that the effect of neuraminidase on the choline uptake were either mediated through glycoproteins or through gangliosides other than those which bind to tetanus toxin (GD1b and GT1b). Several speculative models for explaining the effect of neuraminidase on choline uptake are proposed.

Sulpholipid metabolism in developing chicken retina.

Glycolipid analysis of chicken retina and brain indicated the presence of cerebroside, cerebroside 3‐sulphate and sulphogalactosylglycerolipid In retina, the ratio of cerebroside to cerebroside 3‐sulphate was approximately half compared to brain. During chicken retina ontogenesis the ratio of cerebroside 3‐sulphate to sulphogalactosylglycerolipid increased rapidly and in the adult animal, the amount of cerebroside 3‐sulphate was 14 times higher than that of sulphogalactosylglycerolipid.