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Recently, we investigated the effects of eicosapentaenoic acid (EPA), a fatty acid which modulates immune response and stimulates myelin gene expression, in an established model of multiple sclerosis (MS): the experimental autoimmune encephalomyelitis (EAE) induced in Dark Agouti rats. As scientific evidences and our previous studies have suggested that EPA could directly affect oligodendrocytes, we have now evaluated the effects of EPA in the non-immune mediate MS model characterized by selective oligodendrocytes damage induced by cuprizone (CPZ). We found that feeding weanling rats diets containing 0.6% CPZ for 2 weeks induced variation of whole brain and myelin biochemical composition representative of a severe myelin damage. We thus administered daily and by gavage EPA or PBS to 2-day old rats up to 21 days. Afterwards, rats were fed CPZ diet for 9 days. The results show that compared to PBS/CPZ fed rats, the whole brain cerebroside content in EPA pre-treated rats was statistically increased as well as there was an overall trend of increase of all other biochemical components., (© 2013 Published by Elsevier Ltd.)

The function of PMP70, one of the four ABC half-transporters of mammalian peroxisomes, encoded by ABCD3 gene, is still unclear. The finding that PMP70 over-expression partially corrected very long-chain fatty acid oxidation defects in fibroblasts of X-linked adrenoleukodystrophy patients, has unveiled its potential clinical relevance, prompting us to set up a model system to study PMP70 function. We used the RNA interference technique, a powerful approach to loss-of-function gene expression analysis, to knockdown the ABCD3 gene in the rat glial C6 cell line, since glia could represent the target tissue of X-linked adrenoleukodystrophy disease. Cells were transfected with a vector for RNA interference generating small interfering RNAs that specifically target the ABCD3 mRNA. By using a puromycin-selectable version of the plasmid, we generated a stable cell line (abcd3kd), in which we observed a stable decrease of PMP70 protein expression greater than 70%. We thus examined the effect of ABCD3 knockdown on lignoceric and palmitic acids beta-oxidation and we found that in abcd3kd cells the rate of peroxisomal and mitochondrial beta-oxidation activities were both reduced about one-third compared with control cells. The mitochondrial membrane potential, determined by cytofluorometric analysis, was also affected. Lipid and fatty acid analyses of abcd3kd cells showed an increase of hexacosenoic acid (C26:0) in the cholesteryl-ester fraction. These results add another clue about the overlapping function of PMP70 and ALDP, the peroxisomal protein involved in X-linked adrenoleukodystrophy, since C26:0 is the biochemical marker of the disease and in the brain lesions it is accumulated in the cholesteryl-ester fraction. Considered as a whole, our results indicate that the abcd3kd cell line is a valuable tool to further study the function of PMP70 and eventually its role in X-linked adrenoleukodystrophy.

X-linked Adrenoleukodistrophy (ALD) is characterized by an increase of very long chain fatty acids (VLCFA) in particular of hexacosanoic acid (HA), in tissues and fluids. The biochemical abnormality is due to the dysfunction of peroxisomal degradation of VLCFA. To-date it is unclear if the demyelination which characterizes this disease is the direct consequence of HA accumulation. In order to investigate whether the large amounts of exogenous HA could affect myelin synthesis, 500 micrograms of this fatty acid dissolved in peanut oil were administered daily and by gavage to newborn rats. Since myelin is actively synthesized during early neonatal life and it can be altered by environmental factors including diet, we analyzed lipid and protein composition of myelin after 20, 30 and 60 days of HA administration. Our results show that exogenous HA is incorporated in myelin where it determines biochemical alterations in normal rats having a functioning peroxisomal system. Even though the differences between controls and treated rats are slight, we observed in test rats, a decrease of 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity and of myelin basic protein (MBP) content at any time studied. The decrease of glycolipids (GL) was present only after 20 days of treatment. Since these parameters are related to myelin development, our data lead us to think that the myelin of the treated animals is less mature than that of controls.

Myelinogenesis is a scheduled process that depends on both the intrinsic properties of the cell and extracellular signals. In rat brain, myelin development is an essentially postnatal event and environmental interferences could affect myelin synthesis. Nutrition plays an important role, since severe postnatal malnutrition and essential fatty acid (EFA) deficiency cause hypomyelination. Even though the dietary effects are more pronounced in the postnatal period, dietary lipids can affects myelin development also in the postweaning period. Rats fed with diets rich in polyunsaturated n3 fatty acids showed a decrease of the relative amount of myelin basic protein (MBP) and a CNPase activity indicating a delay in myelin deposition and/or an instability of its structure. Our recent studies have shown that dietary fatty acids can be positively involved in the control of central nervous system (CNS) myelinogenesis. Offspring of rats fed diets containing odd chain fatty acid during pregnancy and lactation show an early development of behavioral reflexes linked to myelination compared to controls fed a diet containing margarine. Subsequent studies have shown that the expression of myelin proteins is higher in test than in control animals, but the mechanism of the action of fatty acids is still unknown. Also human brain myelinogenesis can be affected by environmental factors. EFA deficiency has been well studied for the important role of C22:6 (a C18:3 metabolite) in the vision system development. The observation that dietary fatty acids can affect membrane composition has led to the use of modified diets in some CNS pathological conditions. For example, preterm infants characterized by low levels of C22:6 and fed with formulae diets enriched in this fatty acid, show a recovery of visual function. The administration of C22:6 has also been tested in patients affected by peroxisomal biogenesis disorders which are associated with very low levels of this fatty acid in the brain. During the treatment, C22:6 content increases in red blood cells, and probably in the brain membranes, as considerable neurologic and electrophysiological improvement suggest. A mixture of glyceryltrierucate and glyceryltrioleate has been tested in the demyelinating disease Adrenoleukodistrophy which is characterized by an abnormal accumulation of very long chain fatty acids (VLCFA) in tissues and fluids. The diet is able to lower VLCFA levels in plasma, but its efficacy for myelin damage is debated. Lastly, a diet which reduces the intake of saturated fatty acid and increases the quantity of polyunsaturates is suggested for multiple sclerosis patients since a decrease of linoleic acid in their plasma and erythrocytes has been observed. Such a diet seems able to reduce the severity of the attacks.

Our previous work showed an early development of behavioral reflexes in rats whose mothers had been fed, during pregnancy and lactation, a lipid fraction extracted from yeast grown on n-alkanes (which contain 50% odd-chain fatty acids) in comparison with controls fed a margarine diet. To clarify whether the observed changes might be linked to an early myelination, we have investigated mRNAs involved in myelin synthesis in the brains of offspring at 5 days of age by northern blot and in situ hybridization. Northern blot analysis showed that proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) mRNAs were higher in animals on the lipid diet compared with controls. In situ hybridization with probes specific for PLP, myelin basic protein, and MOG mRNA showed significantly higher numbers of positive cells in test animals compared with controls in all brain regions. This study shows an acceleration of myelinogenesis induced by dietary lipids. These data can give a new insight in the therapeutical approaches involved to promote repair in demyelinating diseases.

The effect of hypothyroidism on the lipid composition of myelin and synaptosomes isolated from adult rat brain was investigated. The animals were made hypothyroid by adding 0.05% propyl-2-thiouracil to their drinking water for four weeks. This pathological state resulted in a significant increase in the relative percentage of choline glycerophospholipids in synaptosomes with a concomitant decrease in ethanolamine glycerophospholipids as compared to controls. In myelin, hypothyroidism significantly influenced only the relative percentage of sulfatides. The effect of the hypothyroid state on mature brain was also reflected in changes in the membrane fatty acid composition. Myelin and synaptosomes showed an increase in arachidonic (20:4) and eicosatrienoic (20:3) acids and an increase in the fatty acid unsaturation index. Furthermore, the 20:4/20:3 and 20:3/18:2 ratios were lower and higher, respectively, in treated animals. The data indicate that hypothyroidism affects the lipid composition of synaptosomes and myelin even though the effects were less pronounced in myelin. The lipid changes observed in hypothyroidism may be of physiological significance, as it is well known that lipid composition modulates various membrane-bound enzymes, transporters and receptors.

Our previous work has shown an early development of behavioral reflexes in the offspring of rats maintained on diets containing a lipid fraction extracted from yeast (Candida lypolitica) grown on n-alkanes during pregnancy and throughout lactation. Since some of these changes could be linked to an early myelination, in this study we investigated myelin maturation in the rat brain by immunohistochemistry. At 7 days the test groups showed considerable immunopositivity to myelin basic protein and proteolipid protein at a more rostral level, such as the corpus callosum where immunopositivity is usually detected later in brain development. At 7 days in controls, staining fibers were detected only in the lower brainstem and in the cerebellum. Immunopositivity in the same regions was more intense in the test groups. Some litters were fostered at birth to produce 2 groups of animals: pups whose mothers were fed a control diet prenatally and the test diet postnatally, and vice versa. Positive fibers are already present at 7 days in the telencephalon area in both groups. These data indicate that dietary lipids can interfere with brain development by accelerating myelinogenesis.

The metabolism of sphingolipids in the central nervous system (CNS) has been studied in adult rats by intraventricular administration of fluorescent ceramide (CER). Rats were sacrificed at various time points post inoculation and the fluorescence of CER, cerebrosides (CB), sulfatides (SULF) and sphingomyelin (SPM) was determined in the CNS myelin and in the pellet, containing mainly microsomes, obtained by Norton myelin preparation. The incorporation of fluorescence was more in the pellet than in the myelin at all times studied. Initially the fluorescence present in the pellet was prevalently due to untransformed CER but an increase of fluorescent products with time was observed. CB was the main product up to 2 h post inoculation, then it decreased with concomitant increase of fluorescent SULF. In the myelin we did not observe differences in incorporation and transformation of fluorescent CER with time: CB was the main fluorescent product at all times studied. At 0.5 h post inoculation the fluorescence, observed by fluorescence microscope, was located in the cell lining the ventricles while after 24 h it appeared also in the paraventricular areas.

Lipid composition of myelin fractions isolated from Lewis rats during the early stage of the development of experimental allergic encephalomyelitis (EAE) were determined by high-performance thin layer chromatography (HPTLC). When comparing the myelin fractions of EAE-affected animals with those of controls, the main differences were observed in the light fraction, where a decrease in the percentage of phospholipids (PH) relative to the total lipids was observed. These findings give further support that the light myelin fraction being the most sensitive at the onset of clinical symptoms must play a key role in demyelinating process.

Biochemical studies of myelin fractions were undertaken on Lewis rats during various time-points in the development of chronic-relapsing experimental allergic encephalomyelitis (CR-EAE). Lipid and protein composition of myelin fractions obtained by sucrose density gradient centrifugation at 10, 19, 24, and 66 d postinduction (pi) were determined by high-performance thin-layer chromatography (HPTLC) and sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS PAGE), respectively. When comparing the myelin fractions of CR-EAE affected animals with those of controls, main differences were observed at 10 d pi. These changes were particularly evident in the light myelin fraction, where a decrease in the percentage of phosphatidylethanolamine and small basic protein relative to the total lipids and proteins of the fraction were observed. At 19 and 24 d pi no biochemical differences were present in both fractions. At 66 d pi, differences in the lipid composition were observed again only in the light myelin fraction. These findings suggest that the light myelin fraction is the most sensitive, particularly at the early stages of the disease, and must play a key role in demyelinating processes.

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of "light", "heavy" and "membrane fraction" lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the "membrane fraction" resembles in its lipid and fatty acid composition other cell membranes (particularly oligodentrocytes); ii) "light" and "heavy" myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nonhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the "heavy" subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.

We have developed a simple method involving high-performance thin layer chromatographic separation of total brain and myelin lipids. Only two solvent systems consisting of chloroform: methanol:acetic acid and water at different concentrations were needed. The plate was then stained with three sequential procedures to visualize phospholipids, cholesterol and galactolipids. Densitometric procedure at each step of staining was utilized to obtain quantitative analysis of brain and myelin samples.

Morphological, biochemical, and physicochemical studies of myelin subfractions were undertaken on the progeny of Sprague-Dawley rats fed diets containing lipids either extracted from yeasts grown on n-alkanes or from margarine. Myelin subfractions obtained from pooled brain homogenates of littermates by sucrose density gradient centrifugation at 7, 14, and 21 days postnatally were subjected to electron microscopy, sodium dodecylsulfate polyacrylamide gel electrophoresis and assayed for 2', 3' cyclic nucleotide 3'-phosphohydrolase activity (CNPase; EC 3.1.4.37). Additionally, surface pressure measurements were made of lipid monolayers derived from myelin subfractions, which were subsequently injected with myelin basic proteins. The myelin subfractions of test animals, when compared with those of controls, show an earlier increase in the specific activity of CNPase, the earlier appearance of low-molecular-weight proteins, and an increase in the affinity of basic proteins for lipids derived from the myelin light fraction. This biochemistry suggests the presence of a more mature myelin between 7 and 14 days in the experimental group. The morphological studies, however, do not seem to concur with the biochemical data. The observed changes are discussed in relation to the influence of dietary lipids on myelinogenesis.

The article presents an abstract of the research paper "Diet, lipids and brain development." Formation of a myelin sheath is one of the fundamental and necessary events in the normal development of the central nervous system in vertebrates. Myelinogenesis is basically a postnatal process in rodents.