Wendy Cammer

Isolation and Characterization of Myelin

In the second edition of the book Neurochemistry, Rossiter (1962) stated, “Since pure ‘myelin’ is not available for direct chemical analysis, the neurochemist has been compelled to deduce the constituents of myelin from such analyses as are practicable.” The available analyses were extensive. Qualitative information had been accumulating from histological staining techniques since the latter half of the nineteenth century. Quantitative studies on brain constituents from which information on myelin could be deduced began shortly after the turn of the century. Thus in his chapter on “The Myelin Sheath, Metabolism of Myelin and Experimental Demyelination,” Rossiter was able to discuss myelin composition and metabolism with considerable assurance and accuracy. The direct analysis of myelin constituents was, however, not possible until techniques were developed for isolating myelin membranes essentially free of other subcellular structures. The first of such procedures were being developed and published in the same year Neurochemistry was published. In this chapter, I will discuss isolation procedures and the composition of purified myelin obtained by these procedures.

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

Abstract: The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white‐matter regions of rat brain, and in Vibratome sections as well as paraffin sections.

A pi form of glutathione-S-transferase is a myelin- and oligodendrocyte-associated enzyme in mouse brain.

Abstract: The pi form of glutathione‐S‐transferase (GST), previously found to be oligodendrocyte associated, has also been found in myelin. In the brains of adult mice, immunocytochemical staining for a pi form of GST was observed in myelinated fibers, as well as oligodendrocytes. In contrast, and as previously found in rats, positive immunostaining for mu forms occurred in astrocytes and not in oligodendrocytes or myelinated fibers. Double immunofluorescence staining strengthened the conclusion that pi was in oligodendrocytes and myelin in mouse brains. The results of enzyme assays performed on brain homogenates and purified myelin indicated that GST specific activities in mouse brain myelin were almost as high (0.8‐fold) as those in mouse brain homogenates. Low, but reproducible, GST activities were also observed in myelin purified from rat brains, in which pi had been demonstrated in oligodendrocytes and mu in astrocytes. The pi form was also stained by the immunoblot technique in myelin purified from mouse brain. It was concluded that pi is a myelin associated, as well as oligodendrocyte associated, enzyme in mouse brain, and possibly also in rat brain. This is the first report of localization of GSTs in mouse brain and of GST in myelin.

Characterization and Localization of Glutathione‐S‐Transferases in Rat Brain and Binding of Hormones, Neurotransmitters, and Drugs

Abstract: Rat brain glutathione‐S‐transferases are rich in Yb type subunits with major RNA transcripts coding for a relatively uncommon Yb3 form. The Yb‐containing isoenzymes of brain cytosol bind glucocorticoids and are covalently labeled with dexamethasone 21‐methanesulfonate. Certain neurotransmitters, hormones, and drugs, such as serotonin, dopamine, glucocorticoids, thyroxine, apomorphine, and benzodiazepine derivatives, are effective inhibitors of brain glutathione transferase activity. Immunocytochemical studies show that Yb forms are localized in ependymal cells, subventricular zone cells, astrocytes, tanycytes, and astrocyte foot processes on blood vessels, but Yb was not detected in oligodendrocytes or neurons. Based on their localization and binding properties, brain glutathione‐S‐transferases have the potential to function in intracellular binding of a variety of compounds and thereby govern their uptake and release in brain, transport to neurons, as well as in their detoxification.

Brain carbonic anhydrase: activity in isolated myelin and the effect of hexachlorophene.

Abstract— Animals receiving hexachlorophene (HCP) in their diet develop cerebral edema with vacuolation of the myelin sheath. When carbonic anhydrase activities were measured in homogenates of brains from HCP‐fed and control rats, the HCP‐fed rats showed small decreases in the enzyme activity, but these changes were not statistically significant. HCP did inhibit the enzyme in vitro but at higher concentrations (10−5‐10−4m) than have been reported for HCP levels in brains of experimental animals. Carbonic anhydrase activity was present in myelin preparations obtained by gradient centrifugation and osmotic shock or by subcellular fractionation. When the latter procedure was used, myelin carbonic anhydrase had a specific activity which was higher than that of the mitochondrial fraction. The myelin enzyme was inhibited by 10−910−8m‐acetazolamide and, like the homogenates and the commercial enzyme, was inhibited by HCP. The mechanism for HCP toxicity remains unknown, but this study does suggest that carbonic anhydrase is an intrinsic component of the myelin sheath.

Differential Localization of Glutathione‐S‐Transferase Yp and Yb Subunits in Oligodendrocytes and Astrocytes of Rat Brain

Abstract: Glutathione‐S‐transferase Yb subunits were recently identified in rat brain and localized to astrocytes, ependymal cells lining the ventricles, subventricular zone cells, and tanycytes. Another isoform, Yp (π family), was detected in rat brain by immunoblotting, and its mRNA was detected by Northern hybridizations. Double immunofluorescence localized Yb and Yp in different glial cells. The strongly Yp‐positive cells were identified as oligodendrocytes by virtue of their arrangement in rows in white‐matter tracts, colocalization in strongly carbonic anhydrase‐positive cells, and association with myelinated tracts in the corpus striatum. Ependymal cells in the choroid plexus and ventricular lining were also strongly Yp positive, whereas Yb was not detected in the choroid plexus. The occurrence of Yp at low levels in astrocytes was indicated after immunostaining by a sensitive peroxidase‐antiperoxidase method, which revealed weak staining of those cells in the molecular layer of the cortex. The data suggest that Yb and Yp subunits are primarily localized to astrocytes and oligodendrocytes, respectively, and that both are absent from neurons. The glutathione‐S‐transferase in oligodendrocytes may participate in the removal of toxins from the vicinity of the myelin sheath. The finding of glutathione‐S‐transferases in ependymal cells and astrocytes in the brain also suggests that this enzyme could be a first line of defense against toxic substances.

Effects of TNFα on immature and mature oligodendrocytes and their progenitors in vitro

Tumor necrosis factor alpha (TNFalpha) appears to take part in the pathogenesis of multiple sclerosis and to contribute to the degeneration of oligodendrocytes as well as neurons. TNFalpha is produced by microglia and astrocytes, which also produce hormones and cytokines that influence its biological activity. Thus, in mixed cultures the effects of exogenous TNFalpha might be modified by products of astrocytes and microglia. The effects of TNFalpha in oligodendrocyte-enriched cultures are reported below. We prepared the cultures by shaking oligodendrocytes off primary mixed glial-cell cultures from brains of 2-day-old rats at 7 days in vitro and plating them (0 days post-shake, DPS). Platelet-derived growth factor and fibroblast growth factor were included in the media at 1-5 DPS in order to encourage proliferation. At 2 DPS media were added with no TNFalpha (controls) or 1000, 2000 or 5000 U/ml of TNFalpha, and at 5 DPS media were replaced with fresh serum-free media. Cultures were fixed with 4% paraformaldehyde at 5, 7, 9 and 12 DPS and immunostained. Oligodendrocyte progenitors were not reduced in numbers immediately after the incubation with TNFalpha (i. e. at 5 DPS). However, after an additional 4 days in culture fewer progenitors remained in the cultures that had been treated with TNFalpha than in the untreated cultures. In the absence of the growth factors there were fewer progenitors, but their numbers also were reduced by TNFalpha. Maturation to the myelin basic protein (MBP)-positive stage was inhibited by about 36% at 9 DPS by 1000-2000 U/ml of TNFalpha, while numbers of O4+/MBP- precursors were unaffected. It is interesting that the steady-state number of O4-positive precursors was unchanged by TNFalpha at 9 DPS, when there were reductions in the numbers of A2B5-positive progenitors and MBP-positive mature oligodendrocytes. That observation suggests that the rates of proliferation, death and maturation are controlled by multiple factors, with a particularly vulnerable time at the maturation to the MBP-positive stage. At 5000 U/ml TNFalpha the specific effect on maturation was overtaken cytotoxicity. These data and a summary of the literature suggest that inhibition of MBP expression is sensitive to lower TNFalpha concentrations and incubation times than is cell survival. Specific effects on numbers of MBP-positive cells, morphology and MBP expression occur at 1000-2000 U/ml for 48-72 h or at up to 10000 U/ml for</=24 h, and the deficits remain after removal of the TNFalpha.

5′‐Nucleotidase in Rat Brain Myelin

Rat brain myelin showed substantial activity of 5′‐nucleotidase. The specific activity in myelin was enriched two‐ to threefold over that in rat brain homogenates, and the total activity in myelin accounted for approximately 24% of the activity in the homogenates. The 5′‐nucleotidase in the homogenates and in isolated myelin had optimum activity at pH 7.5‐9.0, was stimulated by Mg2+ and Mn2+, and was inhibited by Co2+, Zn2+, EDTA, and EGTA. 5′‐AMP, 5′‐UMP, and 5′‐CMP were the preferred substrates, and 5′‐GMP was hydrolyzed at approximately one‐half the rate of the other mononucleotides. The very low rates of cleavage of β‐glycerophosphate and 2′‐AMP ruled out any significant contribution of nonspecific phosphatase to the observed 5′‐nucleotidase activity in myelin. The 5′‐nucleotidase was inhibited by concanavalin A and was protected by α‐methyl‐d‐mannoside against inhibition by that lectin, suggesting that this enzyme in the CNS is a glycoprotein. It is concluded from these data, and from histochemical observations made in other laboratories, that the myelin sheath is one major locus of 5′‐nucleotidase in the rat brain.

Carbonic anhydrase immunostaining in astrocytes in the rat cerebral cortex

Abstract: Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.

The effect of hexachlorophene on the respiration of brain and liver mitochondria

Abstract Hexachlorophene (HCP) in micromolar concentrations increased the rates of respiration of rat liver or calf brain mitochondria utilizing succinate or glutamate plus malate as substrate. Higher concentrations of HCP produced a stimulation of respiration followed by inhibition. When respiration in the presence of ADP had been inhibited by oligomycin, HCP was still capable of stimulating respiration. These observations are typical for uncouplers of oxidative phosphorylation.