Loss of myelin basic protein cationicity in DM20 transgenic mice is dosage dependent.

Demyelination in the transgenic mice depended on the dosage of the cDNA for DM20, in which low copy numbers (two to four and 17 copies of the minigene) showed no signs of demyelination. However when transgenic mice with 17 copies were made homozygous with 34 copies of the DM20 minigene (ND3A hm.) demyelination was observed at around 12 to 16 months compared with ND4 mice having 70 copies of the transgene which had an earlier onset of demyelinating symptoms at 3 months, demonstrating a transgene dosage effect. The process by which demyelination was initiated was associated with changes in myelin basic protein. An increased abundance of less cationic MBP (C-8) isomers occurred prior to demyelination. This increase was also associated with increased activity of peptidylarginine deiminase, the enzyme which converts arginine to citrulline in proteins, thereby providing a mechanism for generating less cationic forms of MBP. These data support a dosage effect of the DM20 transgene.

Myelin basic protein in experimental allergic encephalomyelitis is not affected at the posttranslational level: implications for demyelinating disease.

The microheterogeneity of myelin basic protein, expressed as the ratio between the least cationic (C-8) charge isomer and the most cationic (C-1), was examined in experimental allergic encephalomyelitis (EAE) cases. These included acute EAE of 2 months' duration induced with bovine proteolipid protein in complete Freund's adjuvant (CFA), chronic EAE induced with mouse spinal cord homogenate in varying doses from 0.5 to 2.0 mg in CFA, and chronic relapsing EAE of 12 months' duration induced with synthetic peptide 139-151 of the proteolipid protein sequence. The C-8/C-1 ratio was within the normal range for all groups of animals. However, the C-8/C-1 ratio was six- to sevenfold increased in a spontaneously demyelinating transgenic model, ND4, which contains 70 copies of the cDNA for DM20 (Mastronardi et al.: 1996). Since an increase in the C-8/C-1 ratio was also observed in victims of multiple sclerosis but not other neurological diseases, the ND4 model may address primary changes prior to demyelination, while the EAE model addresses the autoimmune aspects of the disease.

Monoclonal antibodies to proteins of the myelin-like sheath of earthworm giant axons show cross-reactivity to crayfish CNS glia: an immunogold electron microscopy study.

Monoclonal antibodies were generated to the proteins in myelin-like membranes isolated from the nerve cords of the earthworm, Lumbricus terrestris. One of these showing cross-reactivity to 30-32 and 40 kDa proteins was shown by immunofluorescence microscopy and immunogold electron microscopy to be bound primarily to glial cell process and their membranes and the myelin-like layers. This antibody cross-reacted with proteins of 60-65, 42, and 40 kDa in crayfish (Procambarus clarki) nerve cord homogenates. Localization by immunoelectron microscopy showed the antibody to be bound exclusively to the membranes of the glial processes ensheathing the axons in the crayfish nerve cord. Thus, the proteins in earthworm and crayfish glial cell membranes have some epitopes in common. We suggest that this may represent an evolutionary conservation of these proteins.

Modifications of myelin basic protein in DM20 transgenic mice are similar to those in myelin basic protein from multiple sclerosis.

Transgenic mice containing different numbers of transgenes (2-70) of the myelin proteolipid protein DM20 were phenotypically normal up to 3 mo of age, after which the mice containing 70 copies of the transgene spontaneously demyelinated and died at 10-12 mo. Since we demonstrated that demyelination in multiple sclerosis involved specific chemical changes in myelin basic protein (MBP), we investigated the MBP in our transgenic line for similar changes. Both the total amount of MBP in brain and the MBP mRNA levels were unaffected at the different ages. All the isoforms (14-21 kD) of MBP were present, but the microheterogeneity (a posttranslational event) was changed resulting in a higher proportion of the less cationic components reminiscent of the changes in MBP found in multiple sclerosis. An increased amount of the citrullinated form of MBP was found by Western blot analysis. Immunogold labeling of cryosections of brain revealed a greater density of particles with the anticitrulline antibody at 10 mo and that the levels of peptidylarginine deiminase (which deiminates protein-bound arginine to citrulline) were increased. This stable transgenic line represents a useful animal model for the human disease multiple sclerosis.

Locating lipids in the CNS: an historical perspective.

Localization of lipids in the CNS is considered from an historical perspective. General consideration is given to the identification and separation of different parts of the CNS and to the recognition and detection of lipids. Problems associated with each of these aspects are noted. More treatment is given to the localization of gangliosides and the contributions of Leon Wolfe are highlighted.

Demyelination in a transgenic mouse: a model for multiple sclerosis.

A transgenic mouse containing 70 copies (ND4) of the transgene encoding DM20, a myelin proteolipid protein, appeared clinically normal up to 3 months of age. By 8-10 months, it showed tremors, unsteady gait, and died shortly thereafter. We concluded that the clinical symptoms correlated with demyelination based on the following criteria: 1) at 10 months of age only 17% of the amount of myelin obtained from normal mice was isolated from the ND4 mice; 2) astrogliosis, a prominent feature of demyelinating disease was minimal at 3 months of age but prominent by 10 months; 3) at the electron microscopic level disrupted myelin was seen at 8 months of age in the ND4 mice and ingested myelin debris was found in astrocytes; 4) lymphocytic infiltration in association with endothelial cells was observed routinely in the ND4 mice; 5) sections through optic nerves showed denuded and thinly myelinated axons in the 8 month old ND4 mice. Although the mechanism by which demyelination takes place is not fully understood, measurements of the amounts of PLP suggest it is down-regulated by the large amount of DM20. Since DM20 is a major proteolipid in the young but a minor one in the adult, the persistence of high levels in the adult results in improperly assembled myelin which is prone to disruption. Therefore demyelination in the ND4 mouse appears to result from the persistence of immature myelin into the adult.

Characterization of basic proteins from goldfish myelin.

Myelin basic protein (MBP) from common goldfish (Carassius auratus) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130-137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with 125I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microheterogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The metathoracic wing-hinge chordotonal organ of an atympanate moth, Actias luna (Lepidoptera, Saturniidae): a light- and electron-microscopic study.

The structure of a simple chordotonal organ, the presumed homologue of the noctuoid moth tympanal organ, is described in the atympanate moth, Actias luna. The organ consists of a proximal scolopidial region and a distal strand, which attaches peripherally to the membraneous cuticle ventral to the hindwing alula. The strand is composed of elongate, microtubule-rich cells encased in an extracellular connective tissue sheath. The scolopidial region houses three mononematic, monodynal scolopidia, each comprised of a sensory cell, scolopale cell, and attachment cell. The dendritic apex is octagonally shaped in transverse section, its inner membrane lined by a laminated structure reminiscent of the noctuoid tympanal organ 'collar'. A 9 + 0-type cilium emerges from the dendritic apex, passes through both the scolopale lumen and cap, and terminates in an extracellular space distal to the latter. Proximal extensions of the attachment cell and distal prolongations of the scolopale cell surrounding the cap are joined by an elaborate desmosome, with which is associated an extensive electron-dense fibrillar plaque. Within the scolopale cell, this plaque constitutes the scolopale 'rod' material. The data are discussed in terms of both the organ's potential function, and its significance as the evolutionary prototype of the noctuoid moth ear.

Comparative immunohistochemical study of glial filament proteins (glial fibrillary acidic protein and vimentin) in goldfish, octopus, and snail.

Glial fibrillary acidic protein (GFAP) and vimentin proteins are known to be component proteins of glial filaments in the CNS of many vertebrates. The nature of the filaments present in the glial cells of the goldfish optic tectum and in the CNS of two members of the Mollusca (Helix pomatia and Octopus vulgaris) were investigated using immunocytochemical localization of monoclonal antibodies to GFAP and vimentin. Immunoblots visualized by the alkaline phosphatase method showed cross-reactive protein bands to GFAP and vimentin antibodies in total brain homogenates of the goldfish, octopus, and snail. Immunofluorescence staining of the goldfish optic tectum showed GFAP immunoreactivity, primarily in the ependymal cell processes. Immunogold labelling at the ultrastructural level verified that GFAP antibodies were bound to glial filaments. Immunolabelling of the optic lobe of Octopus vulgaris and the cerebral ganglia of Helix pomatia suggests that a protein exhibiting antigenic properties similar to GFAP is a component protein in the filaments of the protoplasmic and filamentous glia randomly distributed throughout the CNS. Unlike anti-GFAP antibodies, which stained relatively specific to filaments, vimentin staining in the CNS tissues of the three organisms studied did not appear to be exclusive to filamentous structures. As vimentin protein has been shown, in previous studies as well as our own, to exist in many tissue types, this suggests that it does not appear to be confined to glial filaments but is shared with other subcellular components. The proteins GFAP and vimentin which are thought to be well conserved in vertebrate evolution also appear to be expressed in the nervous system of some lower organisms.

Isolation and initial characterization of myelin-like membrane fractions from the nerve cord of earthworms (Lumbricus terrestris L).

We report here the isolation of fractions enriched in components of the myelin-like membranes surrounding the giant axons of the earthworm. Lumbricus terrestris L. The composition and purity of the fractions have been assessed using SDS-protein electrophoresis, Western immunoblots, and electron microscopy. Preliminary enzyme assays indicated that the mitochondrial marker, succinate dehydrogenase, has a similar specific activity distribution in earthworm nerve cord and in mouse liver sedimentation velocity fractions, however, the distribution of the total units of activity among the fractions seems to indicate the existence of smaller mitochondria in earthworm nerve cord compared with mouse liver mitochondria. In earthworm nerve cord fractions, Na+/K+ ATPase and Ca2+/Mg2+ ATPase were found to be enriched exclusively in the fraction containing large plasma and myelin-like membranes, while in the mouse liver fractions, the total units of these two enzymes were found to be distributed broadly among fractions. 5'-Nucleotidase activity in the earthworm nerve cord seemed to be restricted to the microsomal fractions (endomembrane network), with a very low activity associated with the large plasma and myelin-like membrane fraction. We have established the presence of keratins or prekeratins in the myelin-like membranes, probably in the form of tonofilaments. However, we could not show that the desmosome-like structures, characteristic of these membranes, are composed of those proteins described for vertebrate epithelial desmosomes.

The use of tetracycline hydrochloride as a rapid fluorescent stain for myelin membranes in vertebrates and invertebrates.

We report here the use of tetracycline as a fast and reliable histological stain of myelinated nerves. These results are significant in that they point to tetracyclines as potentially important probes to study the role of divalent and trivalent cations in myelinated nerves. The usefulness and specificity of tetracycline as a supravital probe for calcium and iron metabolism in myelinated nerves is currently under study.

Effect of acclimation temperature on the axon and fiber diameter spectra and thickness of myelin of fibers of the optic nerve of goldfish.

The optic nerves of common goldfish acclimated to 5 and 25 degrees C were fixed with glutaraldehyde in either phosphate buffer or PIPES with EGTA, post-fixed with osmium tetroxide, and examined by electron microscopy. The axon diameter spectra, from axons measured in electron micrographs and those measured on the electron microscope screen, differ noticeably with acclimation temperature. At the lower temperature, there is a definite shift toward the occurrence of larger fibers compared with the spectrum of the 25 degrees C fish. Although the number of fibers assessed is small compared with the total number in the goldfish nerve, these results confirm our previous study. These findings could be attributed to an increase in the number of new fibers during the acclimation to the higher temperature. We discuss this possibility and on the available evidence find it unlikely. Other changes in the axon and fiber are also seen with acclimation temperature. The axon to fiber diameter ratio, made directly from the electron micrographs, shows that axons from the nerves of the higher acclimation temperature fish possess consistently thicker myelin sheaths than are found for axons in nerves of the lower temperature fish. This finding is also in agreement with results obtained by us from measurements independent of each other.

Effect of acclimation and fixation temperatures on the number of lamellae and periodicity of myelin in fibres of the optic nerve of goldfish.

Previous findings from our laboratory have shown that the optic nerves of goldfish acclimated to different temperatures differ considerably in their glycerophospholipid composition. This paper describes changes in the morphology of the nerve with different acclimation and fixation temperatures. Optic nerves of 5 and 25 degrees C acclimated fish were excised and fixed at the temperature of acclimation, or at the reverse temperature, and the morphology observed by electron microscopy. Under all temperature conditions considered there is a statistically significant linear relationship between the radius of the axon and the number of myelin lamellae. However, the temperature of acclimation and fixation both influence the regression coefficients for this relationship, the higher the acclimation temperature the lower the coefficient and the higher the fixation temperature the higher the coefficient. The periodicity of the myelin also alters with these temperatures, being greater in the 25 degrees C fish than in the 5 degrees C ones. Myelin sheath thickness is also significantly greater in the 25 degrees C fish. These results are discussed in relation to observed changes in glycerophospholipid composition and conduction velocities.

Ca-controlled, reversible structural transition in myelin.

When excised goldfish spinal cord is kept in physiological saline at room temperature, the myelin multilayers swell. As studied by X-ray diffraction, the original repeating distance of 150 A (AS myelin) swells to 177 A (AL myelin); no intermediate distances are seen. At least 70% of the myelin can undergo this gradual conversion. Omitting glucose from the saline, or substituting 2-deoxy-D-glucose for the glucose, or adding NaCN to the saline all promote conversion. Cooling retards the rate but does not prevent conversion. Omitting Ca does prevent conversion, however, and substituting Mg, Sr, or Ba for Ca also does so. Moreover, agents that increase the rate of conversion in Ca-containing saline by up to 5 fold (NaCN, colchicine, A23187) fail to convert myelin in Ca-free saline. We then converted AL myelin back to AS by withdrawing Ca. After converting in NaCN-containing saline, up to 3/4 of the AL myelin recompacts in Ca-free saline; however, none of the myelin recompacts if NaCN is present in the Ca-free saline. Little or no recompaction occurs after conversion in saline without NaCN. Based on our results, we suggest that the oligodendrocytes may maintain AS myelin in vivo by pumping Ca out of the myelin sheath. The myelin in a human PNS nerve has been induced to undergo a similar cycle of swelling and recompaction.

Appearance and phosphorylation of the 210 kDalton neurofilament protein in newborn rat brain, spinal cord, and sciatic nerve.

The appearance and in vivo phosphorylation of the 210 kDalton (kD) neurofilament protein (NF210K) in newborn rat brain, spinal cord, and sciatic nerve were investigated. Electron microscopic examination of neurofilaments isolated from newborn rat brain and spinal cord demonstrated morphologically distinct filaments which contained cross-bridging side arms. Neurofilament proteins, phosphorylated in vivo, were separated by sodium dodecyl sulfate slab gel electrophoresis and were transferred from acrylamide gels to nitrocellulose sheets. The nitrocellulose sheets were treated with antiserum to the 70 kD, 145 kD and 210 kD neurofilament proteins by the immunoblot technique. The three neurofilament proteins were found to be present in newborn brain, spinal cord and sciatic nerve. The presence of NF210K in newborn rat brain was further confirmed by 2-dimensional gel electrophoresis followed by identification of this protein by the immunoblot technique. Exposure of the immunostained nitrocellulose sheets to x-ray film revealed that the NF210K, NF145K, and NF70K proteins were phosphorylated in filaments prepared from newborn rat central and peripheral nervous systems. These results suggest that the synthesis and posttranslational modification of the neurofilament proteins may be synchronized or developmentally regulated. It is feasible that phosphorylation of the NF210K subunit may be a prerequisite for the formation of neurofilament cross-bridging elements which are necessary for radial growth of axons.

The effect of temperature- and oxygen-acclimation on phospholipids of goldfish (Carassius auratus L.) brain mitochondria.

Goldfish (Carassius auratus L.) were temperature- and oxygen-acclimated and the composition of the phospholipids and their acyl groups in brain mitochondria was determined. The proportion of ethanolamine to choline phospholipid was greater while the plasmenyl ethanolamine value (P-GPE/D- + P-GPE) was lower at the low acclimation temperature. For the ethanolamine glycerophospholipids, a rise in the ratio n-6/n-3 fatty acyl groups occurred with cold acclimation. No significant change in the ratio was exhibited by phosphatidyl choline. When the oxygen level was increased, at either acclimation temperature, a rise in the GPE/GPC ratio and the plasmenyl ethanolamine value resulted. The n-6/n-3 ratio was generally increased for the ethanolamine classes when the oxygen concentration was raised. The possible significance of these changes is discussed.

The effects of temperature- and oxygen-acclimation on phospholipids of goldfish (Carassius auratus L.) brain microsomes.

Brain microsome phospholipids and their acyl groups, from temperature and oxygen acclimated goldfish, were investigated. At the lower acclimation temperature (5C) the proportion of ethanolamine- to choline-glycerophosphatides (GPE/GPC) was increased, and the proportion of phosphatidal ethanolamine value decreased. A rise in the n-6/n-3 fatty acyl group also occurred in cold acclimation. Irrespective of acclimation temperature, 25 degrees C or 5 degrees C, a partial replacement of GPC by GPE occurred when the concentration of oxygen was increased; conversely the GPE/GPC ratio decreased at the hypoxic level. The plasmalogen GPE content increased as the oxygen concentration was raised. A rise in the n-6/n-3 ratio, for ethanolamine glycerophosphatides and phosphatidyl choline, occurred when the oxygen concentration was increased (hypoxia to hyperoxia). It is concluded that the lipid alterations associated with thermal acclimation are, in part, attributable to the concomitant change in oxygen concentration.

Immunoblot identification of 13.5 kilodalton myelin basic protein in goldfish brain myelin.

Myelin isolated from goldfish brain shows a multilamellar structure with a major dense line and two intraperiod lines. Sodium dodecyl sulfate gel electrophoresis revealed that the protein profile of goldfish brain myelin is distinctly different from that of rat brain myelin. No protein migrating to the position of proteolipid protein or DM-20 was seen in goldfish myelin. Goldfish acclimated to 5 degrees, 15 degrees, and 30 degrees C showed no qualitative differences in myelin proteins. The 13.5 kD protein in goldfish brain myelin and brain homogenate was intensely immunostained with the antiserum to human basic protein by the immunoblot technique. In contrast, none of the proteins of goldfish myelin were immunostained with antiproteolipid protein serum; however, both proteolipid protein and DM-20 of rat brain myelin were immunostained. The significance of the synthesis of myelin proteins by astrocytes in the goldfish brain is discussed.