Basis for phospholipid incorporation into peripheral nerve myelin

To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23-24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P0 (Golgi) and myelin basic proteins (more direct).     

The regional distribution of myelin oligodendrocyte glycoprotein (MOG) in the developing rat CNS: an in vivo immunohistochemical study

Using an immunohistochemical approach we have characterized the in vivo developmental distribution of myelin oligodendrocyte glycoprotein within the rat CNS. Myelin oligodendrocyte glycoprotein expression emerged in a non-uniform manner during the first 3 postnatal weeks. Although it was absent throughout the CNS of the newborn rat at postnatal day 0(P0), it had appeared in the spinal cord and brainstem by P7. The forebrain and cerebellum remained devoid of immunoreactivity until after P14. Myelin oligodendrocyte glycoprotein emerged at different times within the closely associated fasciculi of the dorsal funiculus. It appeared in the fasciculus cuneatus during the first postnatal week and in the fasciculus gracilis and corticospinal tracts during weeks 2 and 3 respectively. Myelin oligodendrocyte glycoprotein expression developed along a caudo-rostral gradient from spinal cord to forebrain and along an antero-posterior gradient within the CNS in general. The relationship between the onset of myelin oligodendrocyte glycoprotein expression and myelinogenesis was also investigated. In most regions, myelin oligodendrocyte glycoprotein expression lagged behind the initial appearance of myelin basic protein and Luxol Fast Blue-stained myelin by at least 1 week. These observations support the idea that myelin oligodendrocyte glycoprotein is the latest myelin protein to appear in development, only being expressed during the final stages of oligodendrocyte differentiation. Furthermore, the pattern of staggered expression within the dorsal columns indicates that localized, region-specific interactions may comprise a key element in the control of the terminal phases of oligodendrocyte differentiation.     

Biochemical and morphometric analyses show that myelination in the insulin-like growth factor 1 null brain is proportionate to its neuronal composition

To elucidate the role of insulin-like growth factor 1 (IGF1) in the normal development of brain myelination, we used behavioral, biochemical, and histological analyses to compare the myelination of brains from Igf1(-/-) and wild-type (WT) littermate mice. The studies were conducted at postnatal day 40, at which time the Igf1(-/-) mice weighed approximately 66% less than wild-type mice. However, the Igf1(-/-) brain weight was only reduced by approximately 34%. Formal neurological testing showed no sign of central or peripheral myelinopathy in Igf1(-/-) mice. Myelin composition was not significantly different, and myelin concentration, normalized to brain weight or protein, was equal in Igf1(-/-) and WT mice. Likewise, concentrations of myelin-specific proteins (MBP, myelin proteolipid protein, MAG, and 2',3'-cyclic nucleotide,3'-phosphodiesterase) were not significantly different in Igf1(-/-) and WT mice. The myelin-associated lipids galactocerebroside and sulfatide were modestly reduced in Igf1(-/-) brains. Regional oligodendrocyte populations and myelin staining patterns were comparable in Igf1(-/-) and WT brains, with the notable exception of the olfactory system. The Igf1(-/-) olfactory bulb was profoundly reduced in size and was depleted of mitral neurons and oligodendrocytes, and its efferent tracts were depleted of myelin. In summary, this study shows that myelination of the Igf1(-/-) brain is proportionate to its neuronal composition. Where projection neurons are preserved despite the deletion of IGF1, as in the cerebellar system, oligodendrocytes and myelination are indistinguishable from wild type. Where projection neurons are depleted, as in the olfactory bulb, oligodendrocytes are also depleted, and myelination is reduced in proportion to the reduced projection neuron mass. These data make a strong case for the primacy of axonal factors, not including IGF1, in determining oligodendrocyte survival and myelination.     

The dobutamine stress test with thallium-201 single-photon emission computed tomography and radionuclide angiography: postinfarction study

OBJECTIVES: The purpose of this study was to investigate left ventricular wall motion changes during dobutamine-induced myocardial ischemia. BACKGROUND: Dobutamine is increasingly used as a stress test. It has been assumed that high doses of the drug induce the same changes in contractility as physical exercise. However, some data suggest that ischemic myocardium can respond to dobutamine with an increase in contractility. METHODS: Sixty-three postinfarction patients twice underwent the dobutamine test (up to 40 micrograms/kg per min) within 1 to 2 days. Thallium-201 single-photon emission computed tomography (SPECT) and gated equilibrium radionuclide ventriculography were performed on each patient at rest and with dobutamine. Both global and regional ejection fractions were quantified. Sixty patients underwent coronary cineangiography within 1 week. The presence of redistribution was correlated with global and regional ejection fraction changes and with coronary lesions. RESULTS: Redistribution was present in 45 patients, and no change or a decrease in global or regional ejection fraction was detected in 22. In the entire group of patients global ejection fraction increased from 46 +/- 12% to 56 +/- 14%. The six patients with triple-vessel disease had a flat (-0.2 +/- 5%) ejection fraction response to dobutamine, whereas the remaining patients had an increase of 11 +/- 7% (p = 0.003). The regional ejection fraction of the hypokinetic area increased from 27 +/- 10% to 41 +/- 19%, showing no change or a decrease in 13 patients. The 44 patients with peri-infarct redistribution had a significantly higher increase in regional ejection fraction than those without redistribution (16.4 +/- 10% vs. 4.7 +/- 17%, p = 0.003). In the patients with peri-infarct redistribution, an inverse linear correlation was found between redistribution score and dobutamine-induced regional ejection fraction change (r = -0.44, p = 0.004). CONCLUSIONS: Mild to moderate dobutamine-induced peri-infarct ischemia is compatible with an increase in contractility, whereas severe ischemia induces worsening of wall motion.     

Multiple sclerosis: altered expression of 70- and 27-kDa heat shock proteins in lesions and myelin

Recent studies have implicated heat shock proteins (HSP) in the pathogenesis of the multiple sclerosis (MS) lesion. Expression of the 73 kDa constitutive HSP (HSC70), the 72 kDa stress-inducible HSP (HSP70), and the 27 kDa small HSP (HSP27) was analyzed in white matter and myelin from central nervous system (CNS) tissue of MS and normal subjects using a combination of immunocytochemistry and quantitative immunoblotting. Plaques of all types were sharply defined by reduced immunostaining for HSC70, and shown by immunoblotting to contain 30 to 50% less HSC70 than surrounding white matter or normal tissue. In contrast, HSP27 was markedly enhanced 2.5- to 4-fold in plaque regions, especially in fibrous astrocytes and in hyperplastic interfascicular oligodendrocytes at the lesion edge. HSP70 was less abundant than HSC70, and no significant differences in HSP70 levels were noted between MS and normal white matter. Myelin isolated from active plaques contained 3- to 4-fold more HSC70 than normal myelin. Pronounced expression of HSP70 and HSP27 was also found in MS myelin, although neither protein was detected in normal myelin. Thus, white matter undergoing immune-mediated destruction in MS was associated with altered distribution and expression of HSC70 and HSP27. These changes may initially serve to protect myelin from further destruction and facilitate repair; however, enhanced expression of HSC70, HSP70, and HSP27 in myelin may subsequently present as additional immune targets involved in the progression of disease.     

Axonal regeneration and physiological activity following transection and immunological disruption of myelin within the hatchling chick spinal cord

Transections of the chicken spinal cord after the developmental onset of myelination at embryonic day (E) 13 results in little or no functional regeneration. However, intraspinal injection of serum complement proteins with complement-binding GalC or 04 antibodies between E9-E12 results in a delay of the onset of myelination until E17. A subsequent transection of the spinal cord as late as E15 (i.e., during the normal restrictive period for repair) results in neuroanatomical regeneration and functional recovery. Utilizing a similar immunological protocol, we evoked a transient alteration of myelin structure in the posthatching (P) chicken spinal cord, characterized by widespread "unravelling" of myelin sheaths and a loss of MBP immunoreactivity (myelin disruption). Myelin repair began within 7 d of cessation of the myelin disruption protocol. Long term disruption of thoracic spinal cord myelin was initiated after a P2-P10 thoracic transection and maintained for > 14 d by intra-spinal infusion of serum complement proteins plus complement-binding GalC or 04 antibodies. Fourteen to 28 d later, retrograde tract tracing experiments, including double-labeling protocols, indicated that approximately 6-19% of the brainstem-spinal projections had regenerated across the transection site to lumbar levels. Even though voluntary locomotion was not observed after recovery, focal electrical stimulation of identified brainstem locomotor regions evoked peripheral nerve activity in paralyzed preparations, as well as leg muscle activity patterns typical of stepping in unparalyzed animals. This indicated that a transient alteration of myelin structure in the injured adult avian spinal cord facilitated brainstem-spinal axonal regrowth resulting in functional synaptogenesis with target neurons.     

CNP overexpression induces aberrant oligodendrocyte membranes and inhibits MBP accumulation and myelin compaction

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is highly enriched in myelin-forming cells where it is concentrated at the cytoplasmic side of all surface membranes except those of compact myelin. Previous studies have provided evidence that CNP is functionally involved in migration or expansion of membranes during myelination. This hypothesis is supported, in part, by the production of aberrant myelin membranes in transgenic mice that have a 6-fold increase in CNP expression. In addition, many myelin lamellae in these CNP-overexpressing mice lacked major dense lines (MDLs). The purpose of the present study was to determine if CNP overexpression altered: (1) oligodendrocyte and myelin membrane production during early stages of myelination, and (2) the ultrastructural distribution of CNP and myelin basic protein (MBP) in myelin membranes. We identified aberrant membrane expanses that extended from premyelinating oligodendrocyte processes, the periaxonal membrane, and the contact point between oligodendrocyte processes and myelin internodes. Myelin membranes without MDLs were deficient in MBP and enriched in CNP. These data support a functional role for CNP during oligodendrocyte membrane expansion and indicate, for the first time, that CNP may help target MBP to compact myelin.     

Lipid-protein interactions with native and modified myelin basic protein

The basic protein of central nervous system myelin has been shown to form complexes with acidic lipids in vitro. We measured the interaction of myelin basic protein with several charged and neutral lipids in a biphasic chloroform/methanol/water system and investigated the effect of decreasing the electrical charge of the basic amino groups of the myelin basic protein by acetylation. The modified myelin basic protein, which has an average of eight acetyl residues incorporated, was characterised by gel electrophoresis and circular dichroism. Complexes formed between the acetylated myelin basic protein and acidic lipids exhibited a reduction in the amount of lipids bound, a value that could be correlated with the number of modified amino groups. The significance of these experiments with reference to protein-lipid interaction in the myelin membrane is discussed.     

Differences between mentally retarded and normally intelligent autistic children

By means of antibodies labeled with horseradish peroxidase, autologous albumin and IgG were visualized in electron microscopy on the alveolar epithelial surface of the rat lung, as a continuous cell coat, as free granular deposits, or associated with tubular myelin figures. These 2 proteins were detected by immunochemistry in alveolar washing. After ultracentrifugation of alveolar washing, they were still found in the phospholipid-rich precipitate fraction by immunization of rabbits and by immunoelectron microscopy. Thus, albumin and IgG appeared as normal alveolar components in the rat lung; fibrinogen was not found under normal conditions.     

5-Hydroxytryptamine binding to butanol extracts from myelin fragments

The myelin fraction of rat brain stem was treated with butanol-water mixtures, and the extracted proteolipids were separated by Sephadex LH20 column chromatography. 2 peaks of proteolipids eluted in chloroform-methanol 4/1 showed the binding capacity for C14 - 5-HT. This finding suggests the necessity of the more careful investigations for the probability of proteolipids as receptor proteins in the central nervous system.     

Myelin does not influence the choice behaviour of entorhinal axons but strongly inhibits their outgrowth length in vitro

Myelin is crucial for the stabilization of the entorhinohippocampal projection during late development and is a non-permissive substrate for regrowing axons after lesion in the adult brain. We used two in vitro assays to analyse the impact of myelin on rat entorhinohippocampal projection neurons. A stripe assay was used to study the impact of myelin on the choice behaviour of axons from the entorhinal cortex (EC). Given a choice between alternating hippocampal membrane lanes from developmental stages ranging from early postnatal to adult, EC axons preferred to extend on early postnatal hippocampal membranes. Neither the neutralization of myelin-associated factors by a specific antibody (IN-1) nor the separation of myelin from membranes interfered with the axons' choice behaviour. The entorhinal axons showed no preference in the membrane combination of adult and myelin-free adult hippocampal membranes. These stripe assay experiments demonstrate that support for EC axon choice in the developing hippocampus is maturation-dependent and is not influenced by myelin. The application of IN-1 in the outgrowth assay and the separation of myelin from membranes, enhanced elongation of outgrowing entorhinal axons on adult hippocampal membranes, whereas a control antibody did not. This shows that myelin-associated factors have a strong inhibitory effect on the outgrowth length of entorhinal axons. In conclusion, we suggest that axonal elongation in the entorhinohippocampal system during development is strongly influenced by myelin-associated growth inhibition factors and that specific target finding of entorhinal axons is regulated by a different mechanism.     

Fatty acid composition of myelin isolated from the brain of a patient with cellular deficiency of co-enzyme forms of vitamin B12

Myelin fractionation and subsequent lipid isolation have been carried out on a brain from a patient who suffered from a cellular deficiency of the adenosylcobalamin and methylcobalamin co-enzyme forms of vitamin B12. Examination of the fatty acid composition of choline and ethanolamine glycerophospholipids indicated a relative enrichment of odd-chain fatty acids which were identified by gas-liquid chromatography-mass spectroscopy as C15, C15:1, C17 and C17:1. A mixture of methyl branched C17 fatty acids was also identified. Odd-chain fatty acids accounted for 9.8% of the total fatty acid in the myelin choline phospholipid conpared to control values of 1.2%. The affected brain myelin phospholipids had a lower unsaturated fatty acid content. Examination of the myelin sphingolipids, sphingomyelin, cerebroside and sulfatide, yielded abnormal fatty acid profiles. The sphingomyelin contained only small amounts of C24:1 fatty acid. Both normal and hydroxy fatty acid containing cerebroside and sulfatide had reduced levels of C24 fatty acid. Determination of the relative hydroxy and normal fatty acid content of the galactolipids indicated an abnormally high hydroxy fatty acid level. Abnormal fatty acid profiles of brain cerebral sphingolipids have not been previously described in cases of vitamin B12 deficiency. Whether or not these alterations are characteristic will only be established by estimating sphingolipids in other such cases.     

Cerebral blood flow in subarachnoid hemorrhage

Myelin protein zero (MPZ, P0) is well known as the adhesion molecule responsible for the compaction of the myelin sheath of peripheral nerves. Mutations are linked to Charcot-Marie-Tooth syndrome type 1B (CMT1B) and the more severe Dejerine-Sottas syndrome (DSS). Three mutations leading to phenotypes of increasing severity (Ser34del/CMT1B, Ser34Cys/DSS, INS663GC/DSS) were expressed in S2 insect cells and resulted in a decreased adhesion capability in correlation with their respective phenotypes.     

Reevaluation of the growth-permissive substrate properties of goldfish optic nerve myelin and myelin proteins

To determine whether optic nerve myelin of goldfish carries mammalian-like neurite growth inhibitory proteins which can be neutralized by the antibody IN-1, myelin fractions of fish optic nerves were used as substrates for fish retinal ganglion cell axons and rat dorsal root ganglia (DRG). Axonal growth was monitored and compared with that of IN-1 treated preparations. Growth of fish retinal axons and rat DRG neurites was substantial on goldfish optic nerve myelin and no improvement was observed with IN-1. In contrast, rat CNS myelin allowed only poor growth, and number of axons and length of DRG neurites increased significantly with IN-1. In addition, proteins of fish optic nerve myelin and bovine CNS myelin were extracted, reconstituted in liposomes and applied to growth cones. When goldfish myelin proteins in liposomes were seeded onto growth cones, 77% of fish and 89% of rat DRG growth cones continued to elongate, and the proportion of elongating fish growth cones (80%) did not significantly change when liposomes were pretreated with IN-1. But 73% of fish and 93% of rat growth cones collapsed with liposomes containing proteins from bovine CNS myelin. Upon IN-1 treatment, only 24% of fish growth cones collapsed. Thus, axon growth in vitro indicates that goldfish optic nerves, which permit successful axon regeneration in vivo, lack mammalian-like neurite growth inhibitors which are neutralized by IN-1.     

Expression of the apoptosis-effector gene, Bax, is up-regulated in vulnerable hippocampal CA1 neurons following global ischemia

The observation that delayed death of CA1 neurons after global ischemia is inhibited by protein synthesis inhibitors suggests that the delayed death of these neurons is an active process that requires new gene expression. Delayed death in CA1 has some of the characteristics of apoptotic death; however, candidate proapoptotic proteins have not been identified in the CA1 after ischemia. We studied the expression of Bax protein and mRNA, a member of the bcl-2 family that is an effector of apoptotic cell death, after global ischemia in the four-vessel global ischemia model in the rat and compared these results with the expression of the antiapoptotic gene bcl-2. Bax mRNA and protein are both expressed in CA1 before delayed death, whereas bcl-2 protein is not expressed. Bcl-2 protein expression, but not that of Bax, is increased in CA3, a region that is ischemic but less susceptible to ischemic injury. In the dentate gyrus, both Bax and bcl-2 proteins are expressed. The selective expression of Bax in Ca1 supports the hypothesis that Bax could contribute to delayed neuronal death in these vulnerable neurons by an independent mechanism or by forming heterodimers with gene family members other than bcl-2.     

Effect of papain-induced emphysema on the distrubtion of pleural surface pressure

In vivo experiments using 203Pb and radioactively labelled precursors such as [14C] arginine and [3H] tryptophan were performed to identify lead binding components in rat liver. The distribution of lead in 9 tissues and the intracellular distribution in liver and kidney was also investigated. Male rats were injected intravenously with 18 mug of 203Pb/rat and the 203Pb radioactivity was measured in whole tissues as well as in nuclei, mitochondria, lysosomes, microsomes and soluble fractions obtained by centrifugation of liver and kidney homogenates. The subcellular fractions from liver were purified and fractionated into macromolecular components by ultracentrifugation, gel filtration, ion exchange chromatography and solvent extraction. Nuclei were fractionated into membranes, chromatin proteins (histone and residual non-histone proteins) and DNA. Most of the lead was detected in the nuclear membrane fraction bound exclusively to membrane proteins and absent in phospholipids. The intranuclear lead was associated with histone fractions and other basic or very weakly acid proteins as indicated by the incorporation of [14C] arginine and [3H] tryptophan. Lead was present in the chromatographically purified DNA fraction but whether lead was really bound to the nucleic acid was not determined. Mitochondria were fractionated into heavy, soluble and light subfractions representing the inner membranes, the intramitochondrial matrix and the outer membranes respectively. These subfractions contained appreciable quantities of lead. No appreciable lead was present in lipids of the mitochondrial membranes. Significant quantities of lead were associated with the endoplasmic reticulum. Fractionation of microsomes into rough and smooth membranes showed that lead was almost exclusively bound to membranes of rough-surfaced microsomes associated with the heavy rough membrane subfraction. No significant lead was present in the free polysome subfraction or in lipids from the endoplasmic reticulum. More than one lead binding site was identified in the soluble fraction, the high molecular weight components representing the most important lead binding site.     

Secretion and processing of apolipoprotein A-I in the avian sciatic nerve during development

Apolipoprotein A-I (apo A-I), a major apolipoprotein synthesized by liver and intestine to facilitate transport of plasma lipids as lipoproteins, has been detected also in the avian sciatic nerve. The mRNA and protein levels of apo A-I have been shown to increase during the period of rapid myelination (LeBlanc et al.: J Cell Biol 109:1245-1256, 1989). In order to assess the synthesis of apo A-I protein and the processing of apo A-I isoforms during development, endoneurial slices of avian sciatic nerves from chicks during active myelination at 15 and 17 days embryonic and 1 day posthatch age were incubated with [35]S-methionine. The incubations were fractionated into secreted and intracellular fractions, and incorporation of the label was assessed for apo A-I protein. The pattern of labeling of Po protein, as a marker of myelination, was also determined in the intracellular and compact myelin fractions. Methionine incorporation into Po protein was highest in the intracellular compartment at the 15-day embryonic stage and decreased thereafter, with a corresponding increase in the myelin fraction. During these developmental periods, the levels of nascent apo A-I increased in both the secreted and intracellular fractions. The synthesis of apo A-I specifically increases in the secreted fraction compared with total protein synthesis. The processing of the pro-apo A-I is also developmentally regulated. In the intracellular compartment, there are approximately equal proportions of the acidic and basic isoforms. However, with increasing age, a higher proportion of the apo A-I is secreted as acidic isoforms. It is concluded that the secretion and processing of apo A-I is developmentally regulated in the chick sciatic nerve, in parallel with the process of active myelination.     

Translocation of myelin basic protein mRNA in oligodendrocytes requires microtubules and kinesin

Myelin basic protein (MBP) mRNA is localized to the myelin membranes of oligodendrocytes. When exogenous MBP mRNA is microinjected into oligodendrocytes in culture, it is transported along the processes and localized to the myelin compartment in a multistep intracellular RNA trafficking pathway. In the work described here, oligodendrocytes were treated with agents that affect the cytoskeleton including: nocodazole, to disrupt microtubules; taxol, to stabilize microtubules; cytochalasin, to disrupt microfilaments; and kinesin anti-sense oligonucleotide, to suppress kinesin expression. Digoxigenin-labeled MBP mRNA was microinjected into the treated cells and the extent of translocation of the microinjected RNA was determined by confocal microscopy. Nocodazole, taxol, and kinesin anti-sense oligonucleotide inhibited translocation of microinjected MBP mRNA, while cytochalasin B and kinesin sense oligonucleotide did not. These results indicate that translocation of MBP mRNA in oligodendrocytes requires intact microtubules and kinesin but does not require intact microfilaments. The results are discussed in relation to the current multistep model for intracellular RNA trafficking in oligodendrocytes.