The lipid composition of rat brain aggregating cell cultures during development.

The lipid and fatty acid composition of rat brain was studied during its development both in vivo and in an aggregating cell culture system. Although the amount of lipid present in the cultures was very low, the increase in glycolipid content corresponded closely to the period of intense myelin formation. Very long chain fatty acids (hydroxylated and unsubstituted) were present in 41-day cultures. In comparison to the in vivo situation, myelination was delayed in vitro and, after 40 days in culture, cholesterol esters were 5-fold higher than in vivo, indicating that demyelination was occurring.

Infantile form of so-called neuronal ceroid lipofuscinosis: lipid biochemical studies, fatty acid analysis of cerebroside sulfatides and sphingomyelin, myelin density profile and lipid composition.

The biochemical analysis of a case of infantile neuronal ceroid lipofuscinosis, as determined by clinical and neuropathological findings, is presented. A diminished amount of solids is found, the amount of lipids is 30% of the normal as expressed in lyophilized tissue. The yield of myelin isolated by the density gradient is 1.8% of the normal. Phospholipid patterns show a reduction in ethanolamine phosphoglyceride, N-acetylneuraminic acid is extremely low and sphingolipids are largely reduced, cerebrosides being most affected (2.5% of the normal). In cerebrosides and sulfatides the decrease in very long chain fatty acids is important, but the deficiency in any type (including hydroxy compounds) is not too dramatic. According to the aspect under electron microscopy, the density profile, and the biochemical composition of the subfractions, isolated myelin is close to normal. The loss of the myelin sheath appears to reflect a Wallerian degeneration in the CNS: myelin loss is a secondary effect. This disease, from a biochemical point of view, seems to be the ideal control for leukodystrophies.

Is there a blood-brain relationship for saturated fatty acids during development?

Transport of subcutaneously injected [1-14C]-stearic acid through the blood-brain barrier is compared with endogenous biosynthesis (within the brain) during postnatal brain development in mice. The uptake is very important during glial cell multiplication and myelination; endogenous microsomal synthesis is most active during myelination, soluble de novo mechanism is prominent during cell multiplication (mitochondrial systems are not directly related to these events). A parallel is drawn between myelin fatty acids, microsomal synthesis and uptake from the blood.

[Use of a zonal rotor for demonstrating that human myelin is formed by a continuum of particles of different densities]. / Utilisation du rotor zonal pour montrer que la myéline humaine est formée d'un continuum de particules de densités différentes.

Human myelin isolated from frozen brain consists of a continuum of particles as shown by sucrose continuous gradient (between 0,4 and 0,85 M) on zonal rotor. This was determined by measuring the O. D. at 260 and 280 millimicron, the protein concentration by Lowry method and sucrose concentration by polarimetry in 60 fractions. Most of the material is concentrated between 20-23% sucrose, the maximum being found at 21.5% (0.63 M). The fractions are dialyzed for 62 hrs. to remove the sucrose and the pellet obtained after centrifugation shows that the protein concentration increases from the ligher fraction to the heavier.

Pelizaeus-Merzbacher disease: biochemical analysis of isolated myelin (electron-microscopy; protein, lipid and unsubstituted fatty acids analysis).

Analysis of myelin from a leukodystrophic brain was performed (Pelizaeus-Merzbacher disease, classical type). Myelin recovery was 7% of normal, when isolated by ultracentrifugation. Electron microscopy showed a great amount of loose lamellae, with less thick sheaths and periodicity close to normal. This myelin contains fewer lipids than normal, sphingolipids and plasmalogens being reduced. FAtty acids from phospholipids are essentially normal, however enols from plasmalogens are largely reduced. Purified sphingolipids (cerebrosides, sulfatides and sphingomyelin) present a considerable diminution in very long chain fatty acids; the ratio of very long chain fatty acids (over C18) on shorter chains is 1% of the normal value for saturated fatty acids and 2% for the monounsaturated homologues. Protein analysis showed that basic protein and proteolipids were reduced, Wolfgram proteins being relatively increased.

Myelin consists of a continuum of particles of different density with varying lipid composition: major differences are found between normal mice and quaking mutants.

Mouse brain myelin consists of a continuum of particles of different densities, as shown by sucrose density gradient centrifugation. In normal animals most of the material (65 per cent) is concentrated between 0.6 and 0.7 M sucrose (the maximum being found at 0.66 M sucrose, corresponding to 23 per cent). The density differences among various myelin fractions are related to their protein/lipid ratios, as lighter fractions contain less protein and more lipid. Lipid analysis shows a decrease in the amount of every lipid from the lightest to the heaviest fraction: the light fraction is richer in phosphatidyl-ethanolamine, phosphatidyl-serine and cerebrosides. The distribution is highly abnormal in purified myelin from Quaking mutant ; very low quantities of myelin with normal density are found, but unexpected large amount of high density particles are found, possibly related to a "pre-myelin" material (oligodendrogial) processes which are not maturing into normal myelin).

Lignoceric acid biosynthesis in the developing brain. Activities of mitochondrial acetyl-CoA-dependent synthesis and microsomal malonyl-CoA chain-elongating system in relation to myelination. Comparison between normal mouse and dysmyelinating mutants (quaking and jimpy).

Age-related changes in the activities of microsomal and mitochondrial elongating systems have been determined in mouse brain from birth to maturity. In microsomes, the components necessary for behenyl-CoA (docosanoly-CoA) elongation have been found to be NADPH and malonyl-CoA. In mitochondria, both NADH and NADPH are used and acetyl-CoA is the only donor of two-carbon-atoms unit. The synthesised fatty acids were identified by thin-layer and gas chromatography. The specific activity is higher in microsomes than in mitochondria. In microsomes, the specific activity for malonyl-Co-A incorporation reached a maximum at 15 - 20 days of age; this peak was not obtained in the Quaking and Jimpy mutants. The increase in enzyme activity (specific activity and total activity per brain) paralleled the myelin deposition. The activity of the mitochondrial system increases regularly during development: it is not correlated to myelination and it is not affected in the Quaking mutant. The interplay between microsomal and mitochondrial elongation systems is studied.

[Biosynthesis of lignoceric acid in two organelles (mitochondria and microsomes) during the development of the brain in normal and pathologic (Quaking and Jimpy) mice]. / Biosynthèse de l'acide lignocérique dans deux organelles (mitochondries et microsomes) au cours du développement cérébral de la souris, normal et pathologique (quaking et jimpy)

In microsomes, biosynthesis of lignoceric acid from its direct precursor (behenyl-CoA) is largely increased during myelination. The peak is hardly detectable in Quaking; in Jimpy, the synthesis is nearly absent (3% of normal value); in the adult Quaking, the synthesis is normal. Mitochondria are capable of synthesizing lignoceric acid. This synthesis increases regularly during brain development and is normal in both mutants. Saturated fatty acid analysis is brain mitochondria shows that these organelles contain mainly palmitic and stearic acids. However, very long chains are also detectable. Thus it appears that mitochondria synthesize their own acids. Microsomes synthesize their own acids also and myelin fatty acids. There is no interplay between microsomal and mitochondrial metabolisms.

[Comparative biosynthesis of fatty acids, particularly lignoceric acid, in the kidney and brain of normal and "quaking" mice]. / Biosynthèse comparée des acides gras, tout particulièrement de l'acide lignocérique, dans le rein et le cerveau de souris normale et quaking.

Palmityl-CoA elongation is normal in kidney and brain from Quaking mice. However elongation of stearyl-CoA and behenyl-CoA is disturbed in the mutant brain, but not in kidney. Moreove in both organs, the reaction products are the same in normal and Quaking animals. Thus the microsomal biosynthesis of very long chain fatty acid biosynthesis is normal in Quaking kidney and not in brain : the genetic control of elongating enzymes must be different according to the organ.

Nervonic acid biosynthesis by erucyl-CoA elongation in normal and quaking mouse brain microsomes. Elongation of other unsaturated fatty acyl-CoAs (mono and poly-unsaturated).

Biosynthesis of nervonic acid by enzymatic elongation of erucyl-CoA has been studied in mouse brain microsomes. The substrate and cofactor requirements have been measured. Malonyl-CoA and reduced nicotine-adenine-dinucleotide phosphate are required, but not FMN, FAD or NADH. The effect of protein concentration, incubation time, ATP and CoA has been determined; the reaction products were checked by gas-liquid chromatography with automatic counting of the eluate. Very little activity was found in hydroxylated fatty acids. In the presence of phosphotransacetylase (which impedes the de novo microsomal system), the main reaction product was nervonic acid. It is concluded that nervonic acid is biosynthesised by elongation using a two-carbon unit from malonyl-CoA. The same enzyme biosynthesises saturated and mono-unsaturated very long chain fatty acids. The elongation capacity of "quaking" microsomes is reduced to 30% of the normal value with both erucyl-CoA and behenyl-CoA. Elongation of trans isomer (brassidyl-CoA) and poly-unsaturated homologue (clupanodonyl-CoA) was compared to elongation of erucyl-CoA in both normal and mutant mice. Both unsaturated acyl-CoAs are elongated under the same conditions as erucyl-CoA in brain: the poly-unsaturated acyl-CoA is elongated more actively than the mono-unsaturated acyl-CoA in the mutant.

[Substrates and products of fatty acid elongation in mouse brain microsomes]. / Substrats et produits de l'allongement des acides gras dans les microsomes de cerveaux de souris

In brain microsomes, palmitate and stearate elongation involve a membrane lipid-bound substrate. After elongation by malonyl-CoA, acyl-products are partially bound to proteins. Acyl-proteins are not found when endogenous fatty acid elongation takes place. In the dysmyelinating Quaking mouse mutants, "stearyl-membrane" substrate formation is normal; thus, the deficiency observed in very long chain fatty acid formation is not due to a lack in substrate formation.