Joyce A. Benjamins

Metabolism of Myelin

By “metabolism of myelin,” we refer to the molecular events involved in the synthesis of myelin components, and the subsequent assembly, maintenance, and turnover of the myelin sheath. The deposition of myelin involves coordination of the synthesis of its various lipid and protein components, and the interaction of these components to give a stable membrane. Degradation of myelin components occurs as a reaction of the myelin sheath to injury, but may also be required for normal maintenance and remodeling of the membrane. The topographic features of the myelin sheath can be expected to limit partially the metabolism of myelin. The roles of membrane fluidity and cytoplasmic inclusions in the turnover of compact myelin lamellae are not well understood, but obviously metabolism in this system is integrally linked to both anatomical and membrane structure.

Antibody to galactocerebroside alters organization of oligodendroglial membrane sheets in culture

Antibodies to galactocerebroside (GalC) cause major changes in the organization of the membrane sheets elaborated by murine oligodendroglia in culture. Exposure of oligodendroglia to rabbit anti- GalC IgG for 15 min followed by fluoresceinated second antibodies results in patches of surface GalC staining; when second antibodies are applied after 2 hr of anti-GalC, the pattern of staining on membrane sheets is solid and wrinkled. Anti-GalC exposure for 24 hr results in contracted membrane sheets. No membrane contraction is detected in cultures treated with anti-sulfatide IgM or anti-proteolipid protein IgG. In cultures exposed to anti-GalC continuously for 4–7 d, there is a marked decrease in numbers of extended membrane sheets with an accompanying increase in contracted sheets. This effect is reversible upon removal of anti-GalC from the culture media. By scanning electron microscopy, normally flat membrane sheets appear ruffled after 2 hr of anti-GalC treatment; by 24 hr, contracted membrane sheets consist entirely of bulbous protrusions. Oligodendrocyte membranes exposed to anti-sulfatide for 24 hr are not contracted but are covered with bulbous protrusions. The organization of underlying membrane structures was examined in relation to membrane patching and sheet contraction. In membranes with patching induced by exposure to anti-GalC for 15 min, the anti-GalC: GalC complexes are localized over cytoplasmic MBP domains, with the unstained areas located above cytoplasmic microtubular structures. Membrane sheets that are contracted in response to anti-GalC exposure for 6–24 hr show intense GalC staining over microtubular structures. Anti-GalC exposure does not change metabolism of GalC; in cultures incubated with 3H-galactose and anti- GalC for 24 hr, there are no alterations in GalC labeling compared with control cultures. In summary, these results provide direct evidence that interaction between surface glycolipids and external antibodies can initiate a sequence of events leading to dramatic changes within the oligodendrocyte.

Proteins of myelin and their metabolism

The chemical composition of myelin has long been of interest to neurochemists, in part because of the relevance of such information to an understanding of the many human diseases of myelin. In recent years the realization that myelin, although highly specialized in function, shares many of the morphological and chemical properties of other membranes, has focused attention on its suitability as a model system for the study of membrane biogenesis and metabolism. This review stresses recent developments concerning the protein composition of myelin and the metabolism of these proteins.

TNF-α and TGF-β act synergistically to kill Schwann cells

Interactions between cytokines and Schwann cells (SC) are important in development, repair, and disorders of the peripheral nervous system (PNS). Tumor necrosis factor‐α (TNF‐α) and transforming growth factor‐β (TGF‐β) are two prominent cytokines which may be involved in these processes and their gene products are upregulated in some experimental neuropathies. This study focuses on thein vitro effects of these cytokines, both singly and in combination, on cultured SC. Expression of both Type I and Type II TNF‐α receptors was demonstrated on the SC surface by immunocytochemistry. Treatment of SC with a combination of TNF‐α plus TGF‐β causes significant detachment and cell death while treatment with each cytokine alone is not significantly cytotoxic. When compared with control cultures, SC treated with the combination of cytokines exhibit an increase in the number of cells with condensed nuclei and evidence of DNA fragmentation, characteristics consistent with cells undergoing programmed cell death. Thus, TNF‐α plus TGF‐β induce SC loss of adhesion which is predominantly due to cell death. Apoptotic mechanisms are likely to contribute to some extent to this cell death. These findings provide in vitro evidence to support the hypothesis that cytokines can directly damage SC in PNS disorders. J. Neurosci. Res. 53:747–756, 1998.

Secretory products of multiple sclerosis B cells are cytotoxic to oligodendroglia in vitro

B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0-24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35-74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-β1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.

KINETICS OF ENTRY OF PROTEINS INTO THE MYELIN MEMBRANE

Brain slices were prepared from 17‐day old rats, and incubated with [3H]glycine or [3H]‐leucine to label proteins. Myelin was isolated from the slices, and the proteins were separated by discontinuous gel electrophoresis in buffers containing sodium dodecyl sulfate. Radioactive basic and Wolfgram proteins appeared in myelin at similar initial rates, and their entry was nearly linear between 15 and 120 min with no detectable lag. Radioactive proteolipid protein appeared in myelin at one‐fourth the rate of the basic and Wolfgram proteins between 0 and 30 min, then entered at a rate comparable to the other proteins between 45 and 120 min.

Death of Individual Oligodendrocytes in Jimpy Brain Precedes Expression of Proteolipid Protein

Immunocytochemistry and thymidine autoradiography were combined to determine the time elapsed between cell division and the expression of proteolipid protein (PLP) in individual oligodendrocytes in normal mouse brain. In jimpy (jp) brains, autoradiography was used to determine the time elapsed between cell division in an individual oligodendrocyte and evidence of cell death. Oligodendrocytes in normal mouse brain do not express PLP until 72 h after a single injection of [3H]-thymidine. In contrast, oligodendrocytes in jp brains begin to die within 9-11 h after an injection of thymidine. The jp mouse is one of several X-linked, hypomyelinated mutants in which a defect has been demonstrated in the gene coding for PLP. It has been presumed that the lack of this protein in the myelin sheath is responsible for the jp phenotype. However, the present study shows that individual jp oligodendrocytes begin to die long before they would normally have synthesized detectable levels of PLP. Therefore, it seems unlikely that the death of jp oligodendrocytes is due to the absence of PLP in myelin sheaths. Oligodendrocyte death and other early jp abnormalities may be due to the presence of abnormal PLP message which may interfere with glial differentiation. Alternatively, the PLP message may code for another protein which is important for normal development of neuroglia.

Synergism of nitric oxide and iron in killing the transformed murine oligodendrocyte cell line N20.1

Abstract : Nitric oxide (NO) produced in inflammatory lesions may play a major role in the destruction of oligodendrocytes in multiple sclerosis and experimental allergic encephalomyelitis. The transformed murine oligodendroglial line N20.1 is much more resistant than primary oligodendrocytes to killing by the NO generator S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP). This observation prompted investigation of the mechanisms leading to cell death in the N20.1 cells and comparison of SNAP with another NO donor, sodium nitroprusside (SNP). We observed that N20.1 cells were 30 times more sensitive to SNP than to SNAP. The specific NO scavenger 2‐phenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO) protected against SNP only, not against SNAP. However, dithiothreitol protected against both SNAP and SNP, indicating that S‐nitrosylation of cysteines plays a major role in the cytotoxicity of both NO donors. We did not observe any formation of peroxynitrite or increase of Ca2+ concentration with either SNAP or SNP, thus excluding their involvement in the mechanisms leading to N20.1 cell death. Based on two observations, (a) potentiation of the cytotoxic effect of SNP when coincubated with ferricyanide or ferrocyanide, but not sodium cyanide, and (b) protection by deferoxamine, an iron cyanide chelator, we conclude that the greater sensitivity of N20.1 cells to SNP compared with SNAP is due to synergism between NO released and the iron cyanide portion of SNP, with the cyanide accounting for very little of the cytotoxicity. Finally, SNP but not SNAP induces some apoptosis, as shown by DNA laddering and protection by a caspase‐3 inhibitor. These results suggest that low levels of NO in combination with increased iron content lead to apoptotic cell death rather than the necrotic cell death seen with higher levels of NO generated by SNAP.

Effects of monensin on posttranslational processing of myelin proteins.

Abstract: Rat brain slices were incubated with [3H]palmitic acid and [14C]glycine to label the lipid and protein moieties, respectively, of myelin proteolipid protein (PLP). The effects of monensin on posttranslational processing of proteins were examined by measuring the appearance of [14C]glycine‐ and [3H]palmitate‐labeled proteins in myelin and myelin‐like fractions. At 0.01 and 0.10 μM, monensin did not appreciably affect total lipid or protein synthesis; higher concentrations caused increased inhibition. Monensin at 0.10 μM markedly decreased the appearance of [14C]glycine‐labeled PLP in myelin, but had little effect on the 14C basic proteins or the incorporation of [3H]palmitic acid into total or myelin PLP. The same relative effect was apparent at higher monensin concentrations. In the myelin‐like fraction, monensin at 0.10 μM also depressed entry of [14C]glycine into protein comigrating with PLP, and again had no effect on incorporation of [3H]palmitic acid. In addition, monensin increased the [3H]palmitate label associated with two high‐molecular‐weight proteins in the myelin‐like fraction with no concomitant increase in [14C]glycine label.

Cerebroside Sulfotransferase in Golgi-Enriched Fractions from Rat Brain

Abstract: Golgi‐enriched fractions were prepared from brainstems of 17‐day‐old rats by first floating off myelin, then fractionating the remaining pellet by a series of differential and density gradient centrifugations in sucrose. Fractions enriched in Golgi membranes were recovered at 0.46/0.76 m and 0.76/0.87 m interfaces on the final sucrose gradient as indicated by morphology and the biochemical markers thiamine pyrophosphatase and [3H]fucose‐labeled glycoprotein. Morphology of the two fractions indicated very little contamination with myelin lamellae; however, the presence of significant levels of 2′, 3′‐cyclic nucleotidase in the lighter fraction suggested a contribution from oligodendroglial or myelin‐related membranes. Cerebroside sulfotransferase was highly enriched in the lighter Golgi‐enriched fraction relative to the denser fraction, the post‐34, 880 x g microsomes, and the myelin‐like fraction. In contrast, ceramide galactosyl transferase was more evenly distributed among the fractions. Our results show a more highly localized distribution of sulfatide synthesis than of galactocerebroside synthesis, probably in Golgi membranes or oligodendroglia‐related membranes with similar properties.