FATTY ACID COMPOSITION OF CEREBROSIDES, SULPHATIDES AND CERAMIDES IN MURINE LEUCODYSTROPHY: THE QUAKING MUTANT

Abstract— The fatty acid composition of cerebrosides, sulphatides and ceramides has been determined at 20 days postpartum in the brains of Quaking mutant mice and of littermate controls. There was a significant deficit in the proportion of long-chain fatty acids (C₂₂-C₂₄) affecting both normal and a-hydroxy fatty acids of the cerebrosides. The proportion of normal but not the a-hydroxy long-chain fatty acids of the sulphatides was also decreased. Striking and disproportionate deficits of the C_24:1 and C_{24 h:1} fatty acids of cerebrosides, sulphatides and ceramides characterized the brain of the Quaking mutant, and an increased proportion of C_{23 h:O} fatty acid was found in the cerebrosides and sulphatides of the brain of this mutant. We compared these data with findings on the Jimpy mutant which has been examined by the same techniques. The deficiency of long-chain fatty acids which was found in the cerebrosides and sulphatides of both mutants was less extensive but more selective in the Quaking mutant.     

COMPOSITION OF CEREBRAL LIPIDS IN MURINE LEUCODYSTROPHY: THE QUAKING MUTANT

The composition of sphingolipids and phospholipids of mouse brain during myelination was determined in the Quaking mutant, which manifests a genetic disorder of myelin formation, and in littermate controls. The biochemical changes during myelination in the brains of the controls corresponded quantitatively with previous findings in a different strain of mice. The Quaking mutant exhibited concentrations of sphingolipids and phospholipids in brain which were comparable to those of controls in the early stage of myelination but the tissue content failed to increase with maturation. The greatest differences occurred in the cerebrosides which at 65 days of postnatal age were only 10 per cent of control levels. During development the pattern of cerebral levels of sphingomyelin, plasmalogen and total phospholipid in the mutants tended to resemble that of the cerebrosides. The defect in the Quaking mutant is compatible with a failure in maturation of myelin. These findings have been compared with those in the Jimpy mutant, a different genetic disorder of myelin in the mouse previously studied in a similar fashion. The Jimpy mutant is characterized by a quantitatively more pronounced deficiency of myelin lipids and a decline in cerebrosides during brain development.     

Myelin-Deficient Rat: Analysis of Myelin Proteins

Myelin basic protein (BP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity were quantitated in the brains and spinal cords of normal and myelin-deficient (md) rats at 8, 12, 18, and 25 days of age. The levels of BP, MAG, and CNP in 25-day-old md brain were 1.1, 1.8, and 11% of those in controls, respectively. In spinal cord, the levels were higher, at 9, 15, and 12% of control values, respectively. Although BP content in the mutant rats was a lower percentage of the control level than MAG and CNPase contents at all ages, the absolute level of BP increased steadily between 8 and 25 days of age in both brain and spinal cord, whereas there was little change in the amounts of MAG and CNPase during this period. Immunoblotting analysis did not reveal an increased apparent Mr for MAG, as has been observed in quaking and trembler mice. There was little difference in the relative distributions of the 14K, 17K, 18.5K, and 21.5K forms of BP between control and md rat spinal cord homogenates at the ages examined. PLP content was reduced more than that of the other proteins in the md mutants, because it could not be detected by a technique capable of detecting 0.2% of the control brain level and 0.1% of control spinal cord level. This suggests that the expression of PLP may be preferentially affected in the md mutation.     

FATTY ACID COMPOSITION OF CEREBROSIDES, SULPHATIDES AND CERAMIDES IN MURINE SUDANOPHILIC LEUCODYSTROPHY: THE JIMPY MUTANT

The fatty acid composition of cerebrosides, sulphatides and ceramides was determined at 15-16 days post partum in the brain of the Jimpy mutant and in littermate controls. There was a marked deficit in the long chain fatty acids (C₂₂-C₂₄) of cerebrosides and sulphatides of Jimpy brain, with the unsubstituted fatty acids affected more than the alpha-hydroxy fatty acids. A decrease of long chain normal fatty acids was also found in the ceramides of Jimpy brain. The deficit of long chain fatty acids in these sphingolipids of the Jimpy brain was more severe than that found in the Quaking mutant which has a less extensive disorder of myelin formation.     

NEUROTOXICITY OF A NON-METABOLIZABLE AMINO ACID, 1-AMINOCYCLOPENTANE-l-CARBOXYLIC ACID: REGIONAL PROTEIN LEVELS AND LIPID COMPOSITION OF NERVOUS TISSUE

Abstract— The non-metabolizable amino acid, 1-aminocyclopentane-l-carboxylic acid (ACPC), when administered to mice, induces primary degeneration of axons in the cerebellum, rostral spinal cord and peripheral nerves. One to 4 weeks after a single intraperitoneal injection of ACPC (0.5–2 mg/g body wt) in adult mice, the fresh and dry weights of brain, cerebellum and spinal cord were reduced compared with those of normal and pair-fed controls. The protein content of all CNS regions, but particularly that of the cerebellum and cervical spinal cord, was lowered in ACPC-treated mice relative to that of normal controls. Sciatic nerve protein was also decreased in mice following 2 mg/g of ACPC. Pair-fed controls exhibited protein deficits in the cerebellum and cervical spinal cord but to a significantly smaller degree. In ACPC-treated mice, the sulfatide content of spinal cord and peripheral nerve was reduced but that of brain was normal. Sphingomyelin levels in these three regions increased except in the brains of mice given 0.5 mg/g of ACPC where the levels fell.
The protein and sulfatide deficits were greatest in the regions which are known to exhibit the highest proportion of degenerating nerve fibers. The correlation of ACPC treatment with protein and sulfatide loss is consistent with the reported disruptive effects of ACPC on protein metabolism and with the involvement of proteins in sulfatide. metabolism. The protein deficits in pair-fed mice are considered in relation to the exacerbating effect of reduced dietary protein intake on ACPC neurotoxicity.     

Quantitative analysis of free sphingoid bases in the brain and spinal cord tissues by high-performance liquid chromatography with a fluorescence detection

The o-phthaldialdehyde precolumn derivatives of psychosine, sphinganine and sphingosine extracted from brain and spinal cord tissues were determined by high-performance liquid chromatography–fluorescence detection. This method was developed with the purpose of detecting an endogenous amount of psychosine, sphingosine and sphinganine using small aliquots of brain tissues and spinal cord in rats. These sphingolipid bases were extracted in various ratios of chloroform–methanol and several pH values. Recovery of the method is about 81% in 12 ng/tube (final volume, 320 μl), 90–95% in 45 ng/tube of sphingosine and sphinganine within 2–12% relative standard deviation. Detection limits of these sphingoid bases were about 0.05 pmol/mg brain tissue. In the forebrain, brainstem and spinal cord of rats at three different ages of postnatal days (PND) 1, PND 13 and 6 months old, the endogenous concentrations of psychosine, sphingosine and sphinganine were determined. From these results, this method is suitable for the determination of sphingoid bases in small aliquot of brain and spinal cord tissues.     

BIOSYNTHESIS AND COMPOSITION OF BRAIN GALACTOLIPIDS IN NORMAL AND HYPOTHYROID RATS

Abstract— Newborn rats were rendered hypothyroid by methimazole treatment. Incorporation of [1-¹⁴C]galactose both in vivo and in vitro into brain cerebrosides of hypothyroid rats was significantly lower than in normals. Biosynthesis of sulphatides was affected by hypothyroidism to a smaller extent than cerebrosides. Assay of cerebroside biosynthesis from [1-¹⁴C]galactose or UDP-[1-¹⁴C]galactose by brain preparations revealed that incorporation of the sugar in both cases is affected to the same extent by methimazole treatment, suggesting that the phenomenon is not due to impairment of the nucleotide biosynthesis. A radioactive galactolipid tentatively characterized as glycerogalactolipid was synthesized in vitro and its biosynthesis was reduced to a large extent in the brain preparations from hypothyroid rats. The fatty acid composition of cerebrosides and sulphatides from the brains of hypothyroid rats was found to be different from that of normal rats. The percentage of normal C₂₄ fatty acids was significantly decreased in the methimazole-treated rats. Brain sphingomyelin fatty acids did not differ between normal and hypothyroid rats.     

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.     

CHARACTERIZATION OF THE FRACTION OBTAINED FROM THE CNS OF JIMPY MICE BY A PROCEDURE FOR MYELIN ISOLATION

Abstract— A subcellular fraction (called the 0·85-fraction) was isolated from the brains of Jimpy mice by a procedure for obtaining myelin of high purity from immature normal brains. The yield of this fraction obtained from 17-day-old Jimpy mice was only 5 per cent of that from age matched controls. In the electron microscope, the O·85-fractions obtained from 9- and 17-day-old control mice showed many multilayered whorls of myelin, whereas the corresponding fraction from the Jimpy mice was free of multilayered structures which could be recognized as myelin. Basic proteins, proteolipid protein and galactocerebrosides could not be detected in the 0·85-fraction from Jimpy mice although they were major components of the 0·85-fractions from both 9- and 17-day-old control mice. The specific activity of 2',3'-cyclic nucleotide 3'- phosphohydrolase in the Jimpy 0·85-fraction was only 15 per cent of the value for controls. These results can be explained either by the 0·85-fraction from Jimpy brain being a very abnormal 'myelin' or by its being primarily non-myelin contaminants. Little or none of the major glycoprotein found in normal myelin fractions was found in the 0·85-fraction from Jimpy brains. This finding is strong evidence indicating that the glycoprotein is closely associated with normal myelin in situ.     

OCCURRENCE OF ALKANES IN BRAIN MYELIN. COMPARISON BETWEEN NORMAL AND QUAKING MOUSE

Alkanes, a new class of neurolipid, were found in mouse brain, the level being reduced in the Quaking mutant. These hydrocarbons are concentrated in myelin; minor amounts being found in microsomes, mitochondria and synaptosomes. The average recovery is 7.1 μg/mg in normal myelin, 2.2 in the Quaking myelin. The distribution pattern of these alkanes was determined by gas liquid chromatography and was found to differ in normal and Quaking myelin; the hydrocarbons consist mainly of n-alkanes ranging from C₂₁ to C₃₂ with even and odd aliphatic chains.     

Neurotropic behaviour of Trichobilharzia regenti in ducks and mice

The bird nasal schistosome Trichobilharzia regenti is a new agent of cercarial dermatitis. Cercariae are able to penetrate the skin of birds and mammals including man. The parasite then attacks the central nervous system. The present study has shown that schistosomula avoid penetration of blood capillaries and enter the peripheral nerves of the legs of mice and ducks as early as 1 day post-infection (p.i.) and 1.5 days p.i., respectively. These peripheral nerves are used as a route to the spinal cord. In the specific host (duck) schistosomula were found in the spinal cord from 2 days p.i. until 15 days p.i. and in the brain from 12 days p.i. until 18 days p.i. In non-specific hosts (mice; inbred strains BALB/c, hr/hr, SCID) living schistosomula were found in the spinal cord from 2 days p.i. until 21 or 24 days p.i. (depending on the mouse strain) and in the brain of two (BALB/c, SCID) of three inbred strains from 3 days p.i. until 24 days p.i. No correlation was found between the infection dose and clinical status of the experimental hosts. A high affinity of schistosomula for the peripheral nerves was also proved in vitro, suggesting a new type of migratory behaviour in schistosomatids.     

The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice

Background

Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.

Methodology/Principal Findings

The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.

Conclusions/Significance

These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.     

BIPHASIC MYELINATION AND THE FATTY ACID COMPOSITION OF CEREBROSIDES AND CHOLESTEROL ESTERS IN THE DEVELOPING CENTRAL NERVOUS SYSTEM OF THE DOMESTIC PIG

Abstract— The chemical composition of four parts of the CNS (cerebrum, cerebellum, brain stem and spinal cord) was determined in 107 pigs at 11 stages of fetal and postnatal development and also in 6 adults. In cerebrum, cerebellum and brain stem, but not in spinal cord, the rate of increase in weight and the rates of change in lipid content slowed down for a period of about 10 days before and after birth. Cholesterol esters and desmosterol were only found in progressively decreasing amounts during the fetal stages of development and together with DNA these were exceptions to the general increases in the tissue concentrations and total amounts of other components during the period studied.
The onset of myelination, as measured by calculated daily increases in tissue contents of cerebroside took place between 70 and 80 days conceptual age and there were two peaks of activity, the first occurring 2 weeks before and the second 3 weeks after birth. Unlike the rate curve for total spinal cord weight the biphasic accumulation of DNA was not synchronous with myelin lipid accretion and the earlier prenatal DNA peak probably denotes proliferation of oligodendrocytes. The two phases of myelination are discussed in relation to an observed generalized pause in development immediately before and after birth.
Fatty acid analysis of cerebrosides indicated that, in spinal cord, chain elongation and desaturation are associated with myelination and continue with increasing activity until maturity. Consequently there was a progressive decrease in the proportion of saturated fatty acids. The fatty acid components of cholesterol esters in the developing pig were shown to be similar to those found during development in the CNS of other species but different from those found in demyelinating conditions.     

INCORPORATION OF LIPIDS INTO SUBCELLULAR FRACTIONS OF BRAIN OF QUAKING MUTANT

The synthesis of lipids and their assembly into subcellular membrane fractions of the myelin deficient Quaking mutant and control brains was studied in 18-, 24- and 41-day-old animals using a double label methodology with¹⁴C and ³H acetate as precursors. As a general procedure, Quaking mutants were injected intracranially with 50 μCi [¹⁴C]acetate and their littermate controls with 300 μCi [³H]acetate. The animals were killed 3 h post-injection, their brains were pooled and subcellular fractions prepared from the common homogenate. An 80-90% decrease in the incorporation of acetate into eleven lipids of myelin in the Quaking mutant was found. This occurred in the face of apparent normal incorporation (relative to microsomes) into lipids of the other main subcellular fractions (nuclear. mitochondrial and synaptosomal) with the exception of decreased incorporation into the myelin-like fraction at 18 and 24 days. Cholesterol and cerebroside were less readily incorporated into Quaking myelin than the other lipids. Although the microsomal synthesis of cholesterol and cerebroside was depressed by about 30% in the Quaking mutant, the incorporation of cholesterol into nuclear, synaptosomal and mitochondrial fractions was unaffected in the mutant. This indicates that sufficient cholesterol is synthesized for the normal assembly of these organelles. In contrast the incorporation of acetate into cholesterol and cerebroside of Quaking myelin was decreased much more than microsomal synthesis. This latter result is consistent with a defect in the process of myclin membrane assembly     

Lipid content in brain and spinal cord during experimental allergic encephalomyelitis in rats

Abstract— The content of cerebrosides, sulphatides, gangliosides, cholesterol and phospholipids was evaluated in the brain and spinal cord of rats during the acute and recovery stages of experimental allergic encephalomyelitis (EAE). During the acute stage there was a significant decrease of sulphatides and gangliosides in spinal cord; in brain, only sulphatides were diminished. In the recovery stage, cerebrosides and gangliosides were decreased in the brain, whereas the lipid content of the spinal cord was similar to that in control animals. Cholesterol esters were detected in the brain and spinal cord during both periods. The results show that the changes are not the same for brain and spinal cord during the acute and recovery stages and that glycosphingolipids from either white or grey matter seem to be preferentially altered.     

ANOMALIES OF MYELIN-ASSOCIATED GLYCOPROTEINS IN''QUAKING MICE

Abstract— Proteins and glycoproteins in a myelin fraction isolated from Quaking mutant mice were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and stained with Fast Green or with periodic acid-Schiff reagents. Double labelling experiments with [³H]fucose and [¹⁴C]fucose were also used to compare glycoproteins in myelin from the mutant mice with those from control mice. In the myelin fraction from the Quaking mice the basic proteins and proteolipid protein were decreased relative to the high molecular weight proteins. Some glycoproteins which are present in small amounts in myelin from normal mice were increased relative to the major glycoprotein in the myelin fraction of the Quaking mice. Furthermore, the major myelin-associated glycoprotein was shifted toward higher apparent molecular weight in comparison with controls of the same age or even with 9-day-old controls. The abnormal glycoproteins in the mutant myelin fraction could be a factor in the impairment of myelination.     

Warfarin administration reduces synthesis of sulfatides and other sphingolipids in mouse brain

The modulation of phosphosphingolipid synthesis by vitamin K depletion has been observed in the vitamin K-dependent microorganism, Bacteriodes levii. When cultured briefly without the vitamin, a reduction occurred in the activity of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthase. In this report, 16-day-old mice were treated with the vitamin K antagonist, warfarin. Brain microsomes from these animals showed a 19% reduction in synthase activity. Mice treated with warfarin for 2 weeks showed a major reduction in sulfatide level (42%), with a lesser degree or no reduction in levels of gangliosides and cerebrosides. In further experiments, mice were treated with warfarin for 2 weeks and a group was then injected with vitamin K1 (aquamephyton) for 3 days. Enzyme activity returned to a normal level within 2-3 days. Sulfatide levels had increased 33% in the vitamin K-injected group and ganglioside levels also increased, where levels of cerebrosides and sphingomyelin declined. Sulfatide synthesis determined by [35S] sulfate incorporation, showed a 52% increase in incorporation following administration of vitamin K for 3 days. These results suggest a role for vitamin K in the biosynthesis of sulfatides and other sphingolipids in brain. This putative role could be by post-translational protein modification analogous to the role of vitamin K in other systems.     

CONGENITAL HYPOMYELINOGENESIS (BORDER DISEASE) OF LAMBS: POSTNATAL NEUROCHEMICAL RECOVERY IN THE CENTRAL NERVOUS SYSTEM

Abstract— In a neurochemical study of experimental Border Disease in lambs it was found that the fresh weights of four parts of the CNS (cerebrum, cerebellum, brain stem and spinal cord) from clinically affected lambs were significantly smaller than those of controls at birth but by 20 weeks of age the cerebrum, cerebellum and brain stem had reached near normal weights. The spinal cord was still considerably smaller, however. Clinical symptoms of the disease (muscular spasms and'hairy'birthcoat) had disappeared during this period, accompanied by a regression in the neurochemical abnormalities seen at birth. Thus the deficiency of myelin lipids was partially made up by the rapid deposition of cerebrosides and by 20 weeks differences in the fatty acid composition of this lipid fraction were no longer apparent. Myelin degeneration as indicated by the presence of elevated levels of esterified cholesterol was apparently absent at 20 weeks of age and this was parallelled by a fall in the level of'anti-myelin'antibodies in the sera of affected lambs. The altered distribution of copper in spinal cord myelin seen at birth had also become nearly normal at the end of the period.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS IN PERIPHERAL NERVE AND SPINAL CORD OF MONKEY

—The isolation of uronic acid-containing glycosaminoglycans from peripheral nerve and spinal cord of monkey was done by combining the cetyl pyridinium procedure and DEAE-Sephadex column chromatography. The constituent analyses of the isolated GAG-fractions indicated that hyaluronic acid, chondroitin-4-sulphate, chondroitin-6-sulphate, heparan sulphate and a testicular hyaluronidase-resistant galactosamine-containing GAG were present in both tissues. Hyaluronic acid was the predominant GAG (63 per cent) in both tissues and its level was much higher than in brain. Chondroitin-4-sulphate constituted 16 per cent in both tissues. The levels of heparan sulphate and hyaluronidase-resistant galactosamine-containing GAG in these tissues were much lower than in brain. The results indicate that the patterns of GAGs in peripheral nerve and spinal cord of monkey are similar but differ from that of brain.     

REGIONAL DISTRIBUTION AND PROPERTIES OF THE GLYCINE CLEAVAGE SYSTEM WITHIN THE CENTRAL NERVOUS SYSTEM OF THE RAT: EVIDENCE FOR AN ENDOGENOUS INHIBITOR DURING IN VITRO ASSAY

—The activity of the glycine cleavage system (GCS) was determined in homogenates from five specific regions of the rat CNS (telencephalon, midbrain, cerebellum, medulla-pons, and spinal cord). An inverse trend was noted between the glycine content and the specific activity of the GCS in the regions. A 25-fold range in the enzyme activities was found between the telencephalon (highest) and the spinal cord (lowest). The properties of the GCS activity in CNS homogenates agreed with those properties previously described for this system in partially purified preparations of liver and brain mitochondria (Kikuchi , 1973; Bruin et al., 1973). Within the CNS homogenates, the liberation of CO₂ from the carboxyl carbon of glycine was quantitatively coupled to the formation of serine. The presence of an endogenous inhibitor(s) within neural tissues was suggested by the non-additivity of the activities when homogenates from the various regions were combined. Moreover, homogenates of CNS tissue inhibited the GCS activity of liver homogenates, and an inverse relationship was found between the level of GCS activity in a given region of the CNS and its ability to inhibit the GCS activity of liver homogenates. This inhibition of liver activity was greatest when liver was incubated with homogenates of spinal cord (86%) and lowest when incubated with homogenates of telencephalon (20%). Because of this endogenous inhibition, the apparent activity of the GCS measured in vitro may not reflect the contribution of this enzyme system in the metabolism of glycine in vivo. Although the significance of this inhibition is not known, a possible role is discussed for the regulation of the levels in glycine and one-carbon pools within the CNS.