STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

Biochemical Abnormalities in Spinal Cord Myelin and CNS Homogenates in Heterozygotes Affected by the Shiverer Mutation

Myelin was purified from the spinal cords of normal mice and mice heterozygous for the shiverer mutation, and measurements were made of the major myelin proteins and lipids and the specific activities of three myelin-associated enzymes. The myelin purified from the spinal cords of the heterozygotes (shi/+) was deficient by 30-40% in yield and had an apparently unique composition. In particular, when compared with normal mouse spinal cord myelin, there were more high-molecular-weight protein, less myelin basic protein, a higher protein-to-lipid ratio, and higher specific activities of 2',3'-cyclic nucleotide-3'-phosphohydrolase (EC 3.1.4.37) and carbonic anhydrase (EC 4.2.1.1) in the myelin purified from the shi/+ animals. These abnormalities were reflected in the composition of shi/+ whole spinal cord, where the protein-to-lipid ratio was intermediate between the respective values for +/+ and shi/shi spinal cords. Whole brains from shi/+ mice showed deficiencies in galactocerebroside and galactocerebroside sulfate and an increase in total phospholipid, and the lipid composition in the brains of the shi/shi mice was similar to that reported for another dysmyelinating mutant, quaking. The findings provide the first values for the lipids in normal mouse spinal cord myelin and show that heterozygotes are affected by the shiverer mutation. The observations imply that there can be considerable deviation from the normal CNS myelin content and composition without apparent qualitative morphological abnormalities or loss of function and that the amount of myelin basic protein available during myelination may influence the incorporation of other constituents into the myelin membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.     

THE EFFECT OF HYPOCHOLESTEREMIC AGENTS ON MYELINOGENESIS

Abstract— Three drugs known to inhibit biosynthesis of cholesterol, Clofibrate, 20, 25-diazacholesterol and AY-9944 were administered by stomach intubation to suckling rats. At weaning the rats were killed and subcellular fractions, including myelin, were prepared from the brains and spinal cords and analysed for sterol content. Central nervous tissue fractions from Clofibrate-treated rats showed some decrease in total sterols, but the sterol species were qualitatively normal. AY-9944 given to rats caused high amounts of 7-dehydro-cholesterol to accumulate in all brain and spinal cord fractions with the highest amounts (32–38 percent of total sterols) in myelin. In diazasterol-treated rats desmosterol reached 48 per cent of the sterols of myelin. A group of rats was allowed to survive after the final drug intake (21 days) and their brain and spinal cord sterol content followed up to 60 days. At 30 days the proportion of dehydrocholesterol or desmosterol comprised over half the total myelin sterol. By 60 days of age the 7-dehydrocholesterol had almost completely disappeared from all fractions while substantial amounts of desmosterol were retained in myelin. Myelination was retarded by treatment with AY-9944 and 20, 25-diazasterol, possibly by the limited amount of sterols available. The metabolism of the abnormal myelin constituents in drug-treated animals is discussed in relation to the molecular structure of the myelin membrane.     

Biochemical Analysis of Myelin Proteins in a Novel Neurological Mutant: The Taiep Rat

Abstract: Hemispheres, spinal cords, and sciatic nerves were taken from taiep, carrier, and control rats at ages ranging from 1 day to 16 months. Absolute myelin yields from CNS taiep tissues peaked at ～2 months and then decreased until they reached a low but stable level. Myelin yield from the affected hemispheres expressed as a percentage of age-matched controls decreased continuously from 2 weeks until it reached a stable level of ～10–15%. The same was true for the spinal cords, but here the myelin yield reached a plateau at a slightly higher percentage of 20–25%. In comparison with control rats, isolated CNS myelin fractions from the affected rats had a greater content of high molecular weight proteins. Western blot analyses of CNS homogenates revealed that myelin basic protein (MBP), proteolipid protein, and 2′,3′-cyclic nucleotide 3′-phosphodiesterase were all present but decreased to levels generally consistent with the deficiencies of myelin. However myelin-associated glycoprotein (MAG) levels always were reduced much more than those of the other three myelin proteins, and at younger ages the apparent molecular weight for MAG was increased in the mutants. Western blot analyses of sciatic nerve homogenates showed that the levels of MBP, MAG, and P₀ were not significantly different in control and mutant animals. These results suggested an early hypomyelination of the CNS, with peak levels of myelin at 2 months, followed by a prolonged period of myelin loss, until a very low but stable myelin level was reached. The consistently greater loss of MAG, in comparison with other CNS myelin proteins, is different from most other hypomyelinating mutants in which MAG is relatively preserved in comparison with the proteins of compact myelin. This might be due to microtubular abnormalities in the taiep mutant interfering with transport of myelin proteins and having the greatest effect on MAG because of its most distal location in the periaxonal oligodendroglial membranes.     

NEUROCHEMISTRY OF THE SPINAL CORD IN CONGENITAL TREMOR OF PIGLETS (TYPE AII), A SPINAL DYSMYELTMOGENESIS OF INFECTIOUS ORIGIN

Abstract— In piglets affected with congenital tremor type AII the CNS was not morphologically underdeveloped; spinal cord weight, total DNA content and fat-free dry matter differed little from control values. However the total lipid extractable from affected spinal cords was only about 63% of values established for normal newborn piglets. In particular, the cerebroside content (a myelin-specific lipid) was reduced to about 30% of the 'normal' value. This was parallelled by the results of an in vitro assay of cerebroside synthesis from [³H]galactose which indicated a metabolic impairment. The altered fatty acid profile of isolatcd cerebrosides further suggested a derangement of fatty acid synthesis. Unlike the spinal cords of normal newborn piglets, tissues from affected piglets contained significant amounts of cholesterol esters carrying the characteristic fatty acids associated with demyelination. This implied that the reduced quantities of possibly abnormal myelin were unstable. Abnormal swollen oligodendrocytes were commonly present in the spinal cords of affected piglets and this was consistcnt with the observed impairment of myelin formation.     

DNA Changes in Spinal Cords of Rats with Experimental Allergic Encephalomyelitis

DNA levels were measured in the spinal cords of Lewis rats during the development of and recovery from experimental allergic encephalomyelitis (EAE). Spinal cord DNA was first increased 11 days after immunizing the rats with guinea pig myelin and rose to levels four times that of the Freund's adjuvant controls at day 14, then subsided after day 22. Spinal cord DNA was still 150% of control levels 60 days after immunization. These DNA changes were compared with fluctuations in spinal cord acid proteinase in the same animals. Acid proteinase activity in EAE spinal cord increased later than the rise in DNA and attained a level of 170% of control at days 15-17, then subsided. Spinal cord DNA was higher in rats immunized with whole myelin than in those administered equivalent amounts of purified myelin basic protein. Furthermore DNA was higher in spinal cords of rats immunized with a larger dose of myelin (1.0 mg) than with a lower amount (0.5 mg). Various protease inhibitors including pepstatin, nitrophenyl p-guanidino benzoate, polylysine, and dipropionyl rhein, previously shown to protect Lewis rats against EAE, suppressed the increase of DNA in the spinal cord. Measurement of DNA increases in the spinal cord of EAE animals provides a convenient reproducible measurement of the severity of inflammation in the CNS and provides an objective criterion for assessment of the efficacy of various agents screened as possible therapeutic treatment for multiple sclerosis.     

Phospholipid vesicle aggregation induced by human myelin basic protein

Human myelin basic protein isolated from the brains of individuals who died with multiple sclerosis was more potent in inducing the aggregation of egg phosphatidylcholine vesicles than was the basic protein isolated from the brains of normal individuals. The portion of myelin basic protein which bound to egg phosphatidylcholine vesicles was separated from the free protein by sucrose density gradient centrifugation. Similar amounts of basic protein from normal or from multiple sclerosis brains are bound to the lipid and no consistent differences in the N^G, N^G dimethyl-arginine content of the protein fractions have been found.     

Antibody to myelin constituents: A possible factor in induction of cell-mediated demyelination

Results from this laboratory have demonstrated that¹⁴C-labeled myelin opsonized with antibodies raised to purified CNS myelin in rabbit is phagocytized by cultured macrophages in larger amounts than untreated myelin or myelin opsonized with preimmune serum. The cultured macrophages produced high amounts of radioactive cholesterol ester and triglyceride from the antibody-treated myelin while much less was formed from preimmune serum-treated or untreated myelin. Antiserum to galactocerebroside also greatly enhanced the formation of radioactive cholesterol ester, while that to myelin basic protein as well as to other myelin constituents had little or no effect. Serum from Lewis rats with acute EAE 13–14 days after immunization with whole CNS myelin also stimulated radioactive cholesterol ester formation compared to serum from Freund's adjuvant-injected controls (FAC). Serum from EAE rats as a result of myelin basic protein injection was as active as that from rats with whole myelin injection. No galactocerebroside antibody could be demonstrated in the EAE sera, although a strong immunostaining to myelin basic protein and proteolipid protein was demonstrated. IgG prepared from EAE serum also showed stimulatory effects compared to IgG from FAC serum, but much of the activity was lost, and the possibility that other factors may be involved is discussed. These experiments provide evidence that myelin phagocytosis and digestion by macrophages is enhanced by the presence of antibody to myelin. In EAE this antibody may leak into CNS with the breakdown of the blood-brain barrier. A humoral involvement in demyelination in EAE is implicated, and these findings may be extended eventually to the demyelinative mechanism in multiple sclerosis where IgG is found in large amounts in the CNS.Special Issue Dedicated to Dr. Abel Lajtha.     

2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and 5''-Nucleotidase in the Central Nervous System of a Myelin-Deficient Rat Mutant

2',3'-Cyclic nucleotide 3'-phosphodiesterase activity was examined in brains and spinal cords of normal and myelin-deficient Wistar rats. While the activity in normal brains increased from 0.2 mumol/min/mg protein (units) at 6-10 days to 3.5 units at 25 days of postnatal age, the activity in the myelin-deficient rat remained at 0.2-0.3 units over the same period. In spinal cord, the normal activities were 5.7 and 10.9 units at 12 and 20 days, respectively, whereas they declined in the myelin-deficient rat from 1.06 to 0.79 units for the same age points. 5'-Nucleotidase activities in brain and spinal cord were normal in the myelin deficient rat at both ages.     

Role of the immune response in Sindbis virus-induced paralysis of SJL/J mice

The pathologic role of the specific immune and inflammatory responses to viral infections of the CNS was investigated by using mice which are susceptible (SJL/J) and resistant (C57Bl6 and BALB/c) to the development of experimental autoimmune encephalomyelitis (EAE). Intracerebral inoculation of 10(4) PFU of Sindbis virus (SV) into 6- to 8-wk-old SJL/J mice resulted in a severe and sometimes fatal encephalomyelitis. A mild to severe hind leg paralysis was observed around days 6 to 7 postinfection (pi) which closely resembled EAE stages and persisted for up to 8 wk pi. Immunosuppression with cyclophosphamide on day 4 alleviated the severity of this disease. Significant perivascular and parenchymal infiltration was present in the brains and spinal cords of SV-infected SJL/J mice for up to 1 mo. This apparent immunopathologic reaction was found to be a characteristic of SJL/J mice, because infection of 6- to 8-wk-old BALB/c and C57Bl6 mice with SV did not cause paralytic disease. These mice also exhibited a significantly milder cellular infiltrate which was mostly resolved on day 12 to 14 pi. Titers of virus in the brain and spinal cords of mice were comparable with clearance by day 7 pi. SV-specific lymphoproliferation and serum antibody responses were also comparable in all mice. SV-infected SJL/J mice developed antibodies to myelin components as demonstrated in Western blots and responded to myelin basic protein by lymphoproliferation. Lymph node cells from these mice, after in vitro challenge with myelin basic protein, transferred a mild EAE-like disease to naive recipients and potentiated subclinical EAE into a severe disease.     

METABOLIC ACTIVITY OF CNS PROTEINS IN RATS AND MONKEYS WITH EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE)

—The metabolic activity of proteins from myelin and non-myelin fractions of slices of lesions in monkey brains and in spinal cords of Lewis rats with acute experimental allergic encephalomyelitis was investigated using [1-¹⁴C]leucine as a protein precursor. The uptake in vitro of [1-¹⁴C]leucine into the monkey EAE lesions was greatly increased in both the myelin and non-myelin fractions. Similar findings were made in spinal cord slices of the EAE rat with an average specific activity 341 per cent of control measured in proteins of purified myelin and 415 per cent of control in the non-myelin protein. The increased uptake appeared with the onset of paralytic symptoms 10–14 days after injection. The increased uptake did not appear to be a result of an increased amino acid pool size as measured with uniformly labelled l -leucine, valine, arginine and phenylalanine. The increase in specific activity of the myelin protein of the EAE rats was shown to be associated with the peaks characteristic of myelin protein when separated on polyacrylamide gels and the serial slices counted. Most of the radioactivity of both the control and EAE myelin protein migrated with the high molecular weight fraction, and the largest increase in radioactivity in myelin protein appeared in this fraction. Some increase in specific activity was also found in the basic and proteolipid proteins. Four different guinea-pig antigens were used to induce EAE: whole spinal cord, purified basic protein, purified myelin and basic protein + cerebroside. All caused paralytic symptoms and greatly increased incorporation in vitro of [1-¹⁴C]leucine into spinal cord proteins. The incorporation of [1-¹⁴C]leucine into slices of the inguinal and popliteal lymph nodes of the EAE and Freund's adjuvant control rats were measured and compared with the incorporation into the spinal cord non-myelin fractions. The specific activity of lymph node proteins was of the order of 10 × that of the non-myelin protein of the control spinal cord. Invasion of a moderate number of cells of the order of activity of these lymph nodes could account for the large increase in rate of protein synthesis in the EAE nervous tissue. It is concluded that much of the increased protein synthesis could be due to the inflammatory cells, although a small amount of the total increase appears to be associated with myelin protein. Other changes in metabolism of the CNS tissue of the EAE rat include a lower rate of lipid synthesis and a decreased activity of the tricarboxylic acid cycle.     

MYELIN PROTEINS FROM DIFFERENT REGIONS OF THE CENTRAL NERVOUS SYSTEM

—The protein composition of myelin prepared from specific anatomical regions of the bovine brain and spinal cord was studied by a modification of the method of Gonzalez -Sastre (1970). Spinal cord myelin contained lesser amounts of chloroform-methanol soluble protein and proteolipid protein and had a lower activity of the enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase than did myelin from subcortical white matter. There was no difference, however, in the protein composition of myelin from the various levels of the spinal cord. The amino acid composition of both proteolipid and basic protein showed no significant regional differences. Myelin preparations from both brain and spinal cord contained DM-20 protein.     

Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.     

Role of viral persistence in retaining CD8(+) T cells within the central nervous system

The continued presence of virus-specific CD8(+) T cells within the central nervous system (CNS) following resolution of acute viral encephalomyelitis implicates organ-specific retention. The role of viral persistence in locally maintaining T cells was investigated by infecting mice with either a demyelinating, paralytic (V-1) or nonpathogenic (V-2) variant of a neurotropic mouse hepatitis virus, which differ in the ability to persist within the CNS. Class I tetramer technology revealed more infiltrating virus-specific CD8(+) T cells during acute V-1 compared to V-2 infection. However, both total and virus-specific CD8(+) T cells accumulated at similar peak levels in spinal cords by day 10 postinfection (p.i.). Decreasing viral RNA levels in both brains and spinal cords following initial virus clearance coincided with an overall progressive loss of both total and virus-specific CD8(+) T cells. By 9 weeks p.i., T cells had largely disappeared from brains of both infected groups, consistent with the decline of viral RNA. T cells also completely disappeared from V-2-infected spinal cords coincident with the absence of viral RNA. By contrast, a significant number of CD8(+) T cells which contained detectable viral RNA were recovered from spinal cords of V-1-infected mice. The data indicate that residual virus from a primary CNS infection is a vital component in mediating local retention of both CD8(+) and CD4(+) T cells and that once minimal thresholds of stimuli are lost, T cells within the CNS cannot survive in an autonomous fashion.     

Probing the influence of myelin and glia on the tensile properties of the spinal cord

Although glia have been historically classified as the structurally supporting cells of the central nervous system, their role in tissue mechanics is still largely unstudied. The influence of myelin and glia on the mechanical properties of spinal cord tissue was examined by testing embryonic day 18 chick embryo spinal cords in uniaxial tension following disruption of the glial matrix using either ethidium bromide (EB) or an antibody against galactocerebroside (αGalC) in the presence of complement. Demyelination was confirmed by myelin basic protein immunoreactivity and quantified using osmium tetroxide staining. A substantial loss of astrocytes and oligodendrocytes concurrent with demyelination was observed following EB injection but not αGalC injection. No morphological changes were observed following injection of saline or IgG with complement as controls for EB and αGalC. Demyelinated spinal cords demonstrated significantly lower stiffness and ultimate tensile stress than myelinated spinal cords. No significant differences were observed in the tensile response between the two demyelinating protocols. The results demonstrate that the glial matrix provides significant mechanical support to the spinal cord, and suggests that myelin and cellular coupling of axons via the glial matrix in large part dictates the tensile response of the tissue.     

P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System

P2 protein, a myelin-specific protein, was detected immunocytochemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest the the regional variations in the amount of P2 protein are dut to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.     

Biochemical Correlates of Myelination in Brain and Spinal Cord of Mice Heterozygous for the Jimpy Gene

Brain and spinal cord of female mice heterozygous for the jimpy gene were analyzed during development for activity of ceramide galactosyl transferase (CGT) and for levels of myelin basic protein (MBP). CGT activity was low at 13-14 days in brains of heterozygous jimpy females but showed normal levels by 31-36 days, in agreement with our earlier study of this enzyme. In cord, CGT activity was normal or slightly above normal at all ages studied, from 13-14 days into adulthood. In both brain and cord, decreased levels of MBP were observed at 13 days; by 100 days, amounts of MBP approached normal levels. Proven female carriers of the jimpy gene also showed normal levels of CGT activity, MBP, and isolated myelin at 200-250 days of age in both brain and cord. These biochemical findings agree with previous morphologic measurements in cord demonstrating deficits in myelin at early ages but compensation by 100 days. Our results show that compensation occurs earlier in cord than in brain and that levels of MBP show a closer correlation than CGT activity with amounts of myelin, as measured by either morphometric analysis or direct isolation.     

Lipid and Protein Alterations of Spinal Cord and Cord Myelin of Multiple Sclerosis

Abstract: A comprehensive study was carried out to clarify the chemical compositions of spinal cord, cord myelin, and myelin subfractions of multiple sclerosis (MS). The protein compositions of normal-appearing cerebral white matter and cerebral plaque and periplaque tissues were also analyzed for comparison. MS whole cord samples were found to contain higher amounts of water compared with normal samples. The total lipid contents were below normal. Among the individual lipids, cholesterol content remained unchanged, whereas cholesteryl esters appeared increased in MS cords. The acidic phospholipid concentrations were found to be lower than normal. Glycolipids, such as cerebrosides G_M4, G_M1, and G_D1b, which are abundant in myelin, were all decreased. However, the concentrations of G_M3 and G_D3, which are more characteristic of reactive astrocytes, were highly elevated. The total protein content of MS cord samples was decreased, and the decrease was attributable to the loss of myelin proteins as evidenced by the low recovery of myelin. The concentrations of myelin-specific proteins, such as proteolipid protein and myelin basic protein, were significantly reduced. Other changes in the protein compositions included the accretion of two low molecular weight proteins of approximately 11,000 and 12,000, and the appearance of a periodic acid-Schiff-positive protein with the same electrophoretic mobility as the P₀ protein. Analysis of the isolated myelin indicated that it had a grossly normal protein composition. However, the two low molecular weight proteins and the P₀ protein appeared to be enriched in an upper-phase cord subtraction. We attribute the appearance of the two low molecular weight proteins to the breakdown of proteolipid protein and/or myelin basic protein as a result of demyelination, and the appearance of P₀ to the involvement of PNS myelin. The latter finding provides the first biochemical evidence that in MS cord, remyelination can be achieved in part by invading Schwann cells and/or by the small number of Schwann cells that may be present in the cord.     

SPINAL CORD LIPIDS AND MYELIN COMPOSITION IN BORDER DISEASE (HYPOMYELINOGENESIS CONGENITA) OF LAMBS

Spinal cords from clinically affected newborn lambs, each with muscular spasms (‘shaking’) and a ‘hairy’ birth coat, were found to be deficient in DNA and to contain less myelin and various lipid components, suggesting retarded CNS development equivalent to about 124 days conceptual age. Cerebrosides were notably deficient in whole cord and isolated myelin and contained more saturated and less unsaturated fatty acids than normal. The rate of cerebroside synthesis assayed in vitro was enhanced and taken with the very low tissue concentrations this indicated faster cerebroside turnover and a less stable myelin in the spinal cords of lambs affected with Border Disease. Marked decreases in plasmalogen concentrations, the redistribution of phospholipid fractions, the presence of about 8 per cent cholesterol in the esterified form and the characteristic fatty acid composition of these esters strongly suggest that degeneration is concomitant with myelin immaturity. Hypocupraemia, low concentrations of copper in the cerebrum and increased concentrations in spinal cord myelin are additional features of the clinical disease. The latter result may be related to myelin immaturity.